αvβ6 peptide ligands and their uses

ABSTRACT

AVβ6 peptide ligands, functional variants thereof and their nucleic acids encoding them are disclosed with their uses in the treatment and imaging of AVβ6 mediated diseases.

Cross-Reference to Related Applications

This application is a continuation of U.S. patent pplication Ser. No.12/088,998, filed Nov. 25, 2008, which is the U.S. National Stage ofInternational Application No. PCT/GB2006/003673, filed Oct. 3, 2006,which claims priority from United Kingdom Application No. 0520068.8,filed Oct. 3, 2005. The entire disclosure of each of the aforesaidapplications is incorporated by reference in the present application.

FIELD OF THE INVENTION

The present invention relates to αvβ6 peptide ligands, functionalvariants thereof and their nucleic acids encoding them and their uses inthe treatment and imaging of αvβ6 mediated diseases.

BACKGROUND OF THE INVENTION

Integrins are a large family of cell-surface receptors responsible formediating cell-cell and cells-to-extracellular-matrix (ECM) adhesion.There are at least 24 different integrins, each a heterodimer composedof an α and β subunit, whose expression is determined by several factorsincluding tissue, stage of development, and various tissue pathologiessuch as inflammation and cancer. Although they do not possess anyintrinsic enzymatic activity themselves, subsequent to ligand binding,integrins translate extracellular cues into intracellular signals bybringing into juxtaposition a complex of cytoplasmic structural andsignalling molecules that then interact and determine the cell response.As integrins are involved in most elements of cell behaviour includingmotility, proliferation, invasion and survival their roles in diseasehave been widely reported. In fact, some integrins are thought to playan active role in promoting certain diseases including cancer. Forexample αvβ3 has been implicated in promoting the invasive phenotype ofmelanoma and glioblastoma, owing to its multiple abilities includingupregulating pro-invasive metalloproteinases as well as providingpro-migratory and survival signals. As integrin αvβ3 also is upregulatedon endothelial cells of angiogenic blood vessels and may provide similarsignals for the development of neo-vessels in cancer, such data have ledmany pharmaceutical and academic centres to develop antagonists of αvβ3for therapeutic purposes many of which have been peptides orpeptidomimetics. Thus, understanding the structural basis ofintegrin-ligand interaction would aid design of improved integrinantagonists.

αvβ6 is expressed only on epithelial cells. This integrin is involved inboth normal and pathological tissue processes. Thus αvβ6 is upregulatedby epithelial cells during wound healing and inflammation. It is likelythat the ability of αvβ6 to locally activate TGFβ by binding to itsprotective pro-peptide, the latency associated peptide (LAP), explainsthe function of αvβ6 in these transient pathologies. Thus TGFβ cansuppress inflammatory responses and epithelial proliferation suggestingthat αvβ6 serves as a negative control to dampen-down these processes.However, chronic inflammation can lead to an excess of αvβ6-dependentactivation of TGFβ resulting in fibrosis in the lung of experimentalanimals. It is likely that some pathologies that result in fibrosis inhumans may also involve αvβ6-dependent TGFβ activation. Constitutiveαvβ6 over-expression in the skin of mice resulted in chronic woundsappearing on a significant number of transgenic animals. Thus, chronicwounds associated with human diseases (e.g. certain forms ofEpidermolysis Bullosa) may also promoted or exacerbated by upregulationof αvβ6 on the wound keratinocytes.

Recently, it has become clear that the integrin αvβ6 is a major newtarget in cancer. Although αvβ6 is epithelial-specific, it is weak orundetectable in most resting epithelial tissues but is stronglyupregulated in many types of cancer, often at the invasive front. It hasbeen shown that αvβ6 can promote carcinoma invasion by upregulating MMPsand promoting increased motility so that αvβ6 promotes survival ofcarcinoma cells by upregulating Akt. These data suggest strongly thatαvβ6 is actively promoting the invasive phenotype. This suggestion issupported by the recent report showing that high expression of αvβ6correlates with a significant reduction in median survival by coloncancer patients.

αvβ6 has been identified as a receptor for foot-and-mouth disease virus(FMDV) in vitro by binding through an RGD motif in the viral capsidprotein, VP1.

SUMMARY OF THE INVENTION

The present invention arose from work directed to improvingαvβ6-directed therapies, and more particularly to find novel bindingligands, for example which have an increased binding affinity and/orspecificity improving the treatment and imaging of αvβ6 mediateddiseases. These may have major benefits for patients with αvβ6 mediateddiseases such as chronic fibrosis or carcinoma. In particular αvβ6improved antagonists are highly in demand.

Broadly, the present invention is based on the surprising finding thatthe potency of peptide antagonists of αvβ6 depended on the presence ofspecific secondary structures in the peptide antagonists, and inparticular peptides which comprise the sequence motif RGDLXXL/I (SEQ IDNO: 1/SEQ ID NO: 2), wherein LXXL/I is contained within an alpha helicalstructure. While crystal structure analysis of FMDV had previously shownthat the RGD motif was comprised in a G-H lop of the VP1 capsid protein,which is at the tip of a hairpin turn followed by a 3₁₀ helix, there wasno indication that the position of the binding motif within a specificsecondary structure was important for its binding potency. The presentinventors found that the truncated peptides originating from the VP1protein comprising the RGD motif showed increased binding potency andbinding specificity. In particular, the binding specificity and thebinding affinity increased with increasing helical propensity within thebinding region of the peptide. Without being bound by theory it isthought that the α-helix structure within the LXXL motif of the αvβ6binding peptide allows correct orientation of the RGDLXXL (SEQ ID NO: 1)motif to enable hydrophobic side chains to interact with a binding siteon αvβ6. Moreover, the non-covalent contacts between residues in thehelix and residues in the N-terminus stabilize the hairpin structure andthus present the RGD motif favourably for specific binding to αvβ6.

Accordingly, in a first aspect, the present invention provides a peptidecomprising the sequence motif RGDLX⁵X⁶L SEQ ID NO: 1) or RGDLX⁵X⁶I (SEQID NO: 2), wherein LX⁵X⁶L or LX⁵X⁶I is contained within an alpha helicalstructure.

In a further aspect, the present invention provides an isolated nucleicacid molecule that encodes a peptide as defined herein, and anexpression vector comprising the nucleic acid molecule.

In a further aspect, the present invention provides a peptide, nucleicacid molecule or expression vector as defined herein for use in therapyor diagnosis.

In a further aspect, the present invention provides a pharmaceuticalcomposition peptide, nucleic acid molecule or expression vector asdefined herein and a pharmaceutical acceptable carrier.

In a further aspect, the present invention provides a method of treatingan αvβ6 mediated disease or a disease wherein cells overexpress αvβ6comprising administering to a patient in need a therapeuticallyeffective amount of a peptide, a nucleic acid molecule or an expressionvector as defined herein.

In a further aspect, the present invention provides the use of apeptide, a nucleic acid molecule or an expression vector as definedherein for the preparation of a medicament for the treatment of an αvβ6mediated disease or a disease wherein cells overexpress αvβ6. By way ofexample, these disease include chronic fibrosis, chronic obstructivepulmonary disease (COPD), lung emphysema, chronic wounding skin disease(e.g. epidermolysis bullosa) or cancer.

In a further aspect, the present invention provides a method of imagingepithelial cells in the body of an individual, the method comprisingadministering to the individual an effective amount of a peptide asdefined herein and detecting presence of the peptide in the body.

In a further aspect, the present invention provides a method for thediagnosis or prognosis of an αvβ6 mediated disease, the methodcomprising administering to an individual an effective amount of apeptide as defined herein and detecting presence of the peptide in thebody.

In a further aspect, the present invention provides a method ofdelivering a therapeutic active moiety to a αvβ6 expressing cell or atissue containing αvβ6 expressing cells in a patient, the methodcomprising administering a peptide of the present invention.

In a further aspect, the present invention provides a method ofimproving the binding specificity of an αvβ6 binding peptide byincreasing the alpha helical content of the peptide.

Embodiments of the present invention will now be further described byway of example and not limitation with reference to the accompanyingfigures and tables.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. FarUV-CD Spectra of (A) A20FMDV1, (B) A20FMDV2 and (C) A20LAPpeptides in PBS with TFE concentrations between 0-50% (v/v).

FIG. 2. Mean molecular elipticity of (a) A20FMDV1, (b) A20FMDV2 and (c)A20LAP peptides in PBS with TFE concentrations between 0-50% (v/v).

FIG. 3. Schematic of main NOE and ROE contact types, hydrogen bondacceptors and residues giving rise to f restraints for (a) A20FMDV1 (SEQID NO: 7), (b) A20FMDV2 (SEQ ID NO: 8) and (c) A20LAP (SEQ ID NO: 6).

FIG. 4. Sections of 2D NOESY NMR Spectra for A20FMDV1: (a), (d) and (e);A20FMDV2: (b), (e) and (h) and A20LAP: (c), (f) and (i). Spectra (a-c)cover the Ha-Hb region, (d-f) the NH-NH region and (g-I) the NH-aHregion. All chemical shifts are referenced externally to a 100 μMsolution of dimethylsilapetane sulphonic acid (DSS) in PBS/30%(v/v) TFE.

FIG. 5. Calculated structures for A20FMDV1: (a-c); A20FMDV2: (d-f) andA20LAP: (g-i). Ensembles of 40 structures (a), (d) and (e) show allbackbone bonds (residues 1-20); ensembles of 40 structures (b), (e) and(h) show backbone bonds from GLXX to C-terminus to highlight thecalculated convergence on each helix. The bonds coloured red identifythe LXX[L/I]XXX region that was used to fit the ensembles and createdata in Table 2. Ribbon diagrams (c), (f) and (i) are shown of theensemble average structure for each peptide with the RGD motif shown inball and stick. All figures were created in MOLMOL 2k.2 (Koradi et al,1996).

FIG. 6. 1H STD NMR spectra of integrin αvβ6 and peptide A20FMDV2 in thepresence of Ca2+and Mg2+. (a) and (c) are the control spectrum (no STDtransfer showing peptide signals) whereas (b) and (d) are the STDdifference spectrum with 30 ms spin-lock filter. Expansions (c) and (d)have key residue resonances highlighted in the data.

FIG. 7. The absolute STD NMR transfers in between integrin αvβ6 andA20FMDV2 (SEQ ID NO: 8) shown as a percentage on each amino acid peptidein the presence of Ca²⁺ and Mg²⁺.

FIG. 8. Effect of INK and DD19 peptides on αvβ6-dependent adhesion of3T3 B6.19 to LAP. Shown are the peptide concentration of both peptidesplotted against the percentage of cell adhesion.

FIG. 9. Anti-αvβ6 cyclic peptides bind preferentially to αvβ6-expressingcells. Biotinylated A20FMDV2-Cyc2 or a cyclic scrambled version wasadded to A375Ppuro or A375Pb6puro cells. Bound peptide was detected witheither streptavidin-FITC or mouse anti-biotin antibody followed by goatanti-mouse antibody conjugated to Alexa Fluor488 and samples analysed byflow cytometry. Peptide data are in light grey, background(streptavidin-FITC or mouse anti-biotin antibody followed by goatanti-mouse antibody conjugated to Alexa Fluor488 only) is shown inblack. Note that the A20FMDV2-Cyc2 signal is higher on A375Pb6purocells.

FIG. 10. Concentration-dependent binding of biotinylated peptides toA375Pβ6puro and A375Ppuro. Biotinylated peptides DV1217, A20FMDV1,A20LAP and A20FMDV2 were allowed to bind to A375Pβ6puro and A375Ppuro inthe presence of cations (0.5 mM MgCl2, 1 mM CaCl2) and 0.1% sodiumazide. Grey and black solids represent binding of control antibodies,10D5 (anti-αvβ6, grey solids) and non-immune IgG (black solids). Redlines, 10 μM biotinylated peptide; orange lines, 1 μM biotinylatedpeptide; green lines, 0.1 μM biotinylated peptide; blue lines, 0.01 μMbiotinylated peptide; purple lines, 0.001 μM biotinylated peptide. Dataare representative of at least two independent experiments with similarresults.

Table 1. NMR assignment list of observed 1H chemical shifts for A20FMDV,A20FMDV-2 and A20LAP peptides in PBS/30%(v/v) TFE at 10° C. All chemicalshifts are referenced externally to a 100 μM solution ofdimethylsilapetane sulphonic acid (DSS) in PBS/30%(v/v) TFE.

Table 2. List of NOE, hydrogen bond and torsion angle connectivities forA20FMDV, A20FMDV-2 and A20LAP peptides Table 3. Structural Statisticsfor 35 structure ensembles of A20FMDV, A20FMDV-2 and A20LAP peptides

Table 4. Amino acid sequences of the peptides used in the experimentalexamples.

DETAILED DESCRIPTION

αvβ6 Peptide Ligands

The present invention involves the use of peptides ligands comprisingthe sequence motif RGDLX⁵X⁶L (SEQ ID NO: 1) or RGDLX⁵X⁶I (SEQ ID NO: 2),wherein LX⁵X⁶L or LX⁵X⁶I is contained within an alpha helical structure.Unless specified otherwise, amino acid positions herein are numberedfrom N to C-terminus of the peptide.

The term “alpha helical structure” is understood to be a sequentialgroup of amino acids in a peptide that interact with a particularhydrogen bonding pattern and thus define a helical structure. Forexample, the hydrogen bonding pattern in a standard alpha helix isbetween the carbonyl oxygen of residue n and the amide hydrogen ofresidue n+4. For the 3₁₀-helix, this hydrogen bonding pattern is betweenresidues n and n+3 and for a pi-helix it is between residues n and n+5.The number of residues per turn in each alpha-helix is 3.6, 3.0 and 4.4for the standard alpha-helix, 3₁₀-helix and pi-helix respectively.

An alpha helix useful in the present invention may be an alpha helixmimetic as described in WO95/00534. Alpha helix mimetics are alphahelical structures which are able to stabilize the structure of anaturally occurring or synthetic peptide.

The peptides of the present invention may comprise standard helices, or3₁₀ helices or pi helices or any combination thereof. For example, thehelices of the present invention may comprise amino acids that form a“cap” structure, preferably two caps, an N terminal cap and a C terminalcap which flank the helix.

In a preferred embodiment of the present invention, the peptide definedabove comprises the sequence RGDLX⁵X₆LX⁸X⁹X¹⁰ (SEQ ID NO: 3).Preferably, the peptide comprises the sequence RGDLX⁵X⁶LX⁸X⁹X¹⁰Z_(n)(SEQ ID NO: 4), wherein Z is a helix promoting residue and n is anynumber between 1 and 20. Preferably, n is between 5 and 15, even morepreferably n is between 8 and 12. Extension of the helix to includehelical residues in the Z position are preferred embodiments as theyfurther enhance the helix dipole that can also enhance binding to αvβ6.

The peptides of the present invention can also be functional variant ofthe peptides as defined above, including peptides that possess at least70%, preferable 80%, even more preferable 90% sequence identity with thepeptides above, it includes further peptides comprising unnatural ormodified amino acids. Suitable unnatural amino acids include, forexample, D-amino acids, ornithine, diaminobutyric acid ornithine,norleucine ornithine, pyriylalanine, thienylalanine, naphthylalanine,phenylglycine, alpha and alpha-disubstituted amino acids, N-alkyl aminoacids, lactic acid, halide derivatives of natural amino acids, such astrifluorotyrosine, p-Cl-phenylalanine, p-Br-phenylalanine,p-I-phenylalanine, L-allyl-glycine, b-alanine, L-a-amino butyric acid,L-g-amino butyric acid, L-a-amino isobutyric acid, L-e-amino caproicacid, 7-amino heptanoic acid, L methionine sulfone, L-norleucine,L-norvaline, p-nitro-L-phenylalanine, L-hydroxyproline, L-thioproline,methyl derivatives of phenylalanine—such as 1-methyl-Phe,pentamethyl-Phe, L-Phe(4-amino), L-Tyr(methyl), L-Phe(4-isopropyl),L-Tic(1,2,3,4-tetrahydroisoquinoline-3-carboxyl acid),L-diaminopropionic acid and L-Phe(4-benzyl). The peptides may be furthermodified. For example, one or more amide bonds may be replaced by esteror alkyl backbone bonds. There may be N or C alkyl substituents, sidechain modifications or constraints such as disulphide bridges, sidechain amide or ester linkages.

The peptides of the present invention may include both modified peptidesand synthetic peptide analogues. Peptides may be, for example, bemodified to improve formulation and storage properties, or to protectlabile peptide bonds by incorporating non-peptidic structures.

Peptides of the present invention may be prepared using methods known inthe art. For example, peptides may be produced by chemical synthesis,e.g. solid phase techniques and automated peptide synthesisers, or byrecombinant means (using nucleic acids such as those described herein).For example, peptides may be synthesised using solid phase strategies onan automated multiple peptide synthesizer (Abimed AMS 422) using9-fluorenylmethyloxycarbonyl (Fmoc) chemistry. The peptides can then bepurified by reversed phase-HPLC and lyophilized. The peptide may beprepared by cleavage of a longer peptide, e.g. the 5T4 peptide (GenBankAccession No. Z29083). Thus, the peptide may be a fragment of the 5T4sequence. Peptides may be prepared by recombinant expression of thepolynucleotides described herein.

Peptides are expressed in suitable host cells and isolated using methodsknown in the art.

Preferably, X⁵-X⁶ and X⁸-X¹⁰ are helix promoting residues. Preferably,the helix promoting residues are independently selected from the groupconsisting of Glu, Ala, Leu, Met, Gln, Lys, Arg, Val, Ile, Trp, Phe andAsp. The helix promoting residues could be an artificial amino acid or amodified amino acid.

The term “helix promoting residues” includes amino acids with aconformational preference greater than 1.0 for being found in the middleof an α-helix (from Creighton, 1993 and Pace C. N. and Scholtz J. M.(1998), Biophysical Journal, Vol. 75, pages 422-427). However,non-orthodox helix promoting combinations of amino acids are also withinthe scope of the invention if they enhance the specificity and/oraffinity of binding to αvβ6.

By “terminal capping”, we mean the stabilisation of the alpha helixdipole whereby the N-terminal end of the helix is capped by a negativelycharged amino acid like glutamic acid. Likewise the C-terminal may becapped with a positively charged amino acid like lysine. Cappingresidues may adhere to capping rules as defined by Aurora and Rose(Protein Sci. 7(1):21-38; 1998), but non-orthodox capping motifs arealso within the scope of the invention if they stabilize the peptide bystructural interaction.

In a further embodiment, the peptides of the present invention may becyclised. Methods are well known in the art for introducing cyclicstructures into the peptides of the present invention to select andprovide conformational constraints to the structure that result inenhanced stability. For example, a C- or N-terminal cysteine can beadded to the peptide, so that when oxidized the peptide will contain adisulfide bond, generating a cyclic peptide. Other peptide cyclisingmethods include the formation of thioethers and carboxyl- andamino-terminal amides and esters. A number of synthetic techniques havebeen developed to generate synthetic circular peptides (Tam & Lu,Protein Sci., 7(7): 1583-1592, 1998; Romanovskis & Spatola, J. Pept.Res., 52(5): 356-374, 1998; Camarero & Muir, J. Amer. Chem. Soc., 121:5597-5598, 1999; Valero et al., J. Pept. Res., 53(1): 56-67, 1999).Generally, the role of cyclising peptides is two fold: (a) to reducehydrolysis in vivo and (b) to thermodynamically destabilise the unfoldedstate and promote secondary structure formation. There is some potentialimportance with hydrophobic packing of residues N-terminal to RGD alongthe opposite helix face so that the design of residues X5-X6 could alsoenhance specificity.

In a further embodiment of the present invention, the peptide may berepresented by the following formula B_(n)RGDLXXLXXXZ_(m) (SEQ ID NO:5), where residue B is a residue which enhances the hydrophobicinteractions with the helix defined from LXXL and also enhances thehammerhead RGD for binding, and wherein Z is a helix promoting residueand wherein n is a number between 1 and 35 and independently m is anumber between 1 and 35. Preferably n selected so that B is sufficientlylong to facilitate a hydrophobic/non-covalent interacting core. Theexact nature of these residues depends on the general design of theregion, in particular it is preferred to have a mixture of hydrophobicinteractions (from residues such as Val, Ile, Leu) and/or electrostaticinteractions (using Asp, Glu, Lys and Arg together with theircounterpart ion-pair in the now defined X15-X16 positions (in betweenthe two Leu residues in LXXL).

In a further embodiment, the peptide comprises or consists of asequences selected from the group GFTTGRRGDLATIHGMNRPF (SEQ ID NO: 6),YTASARGDLAHLTTTHARHL (SEQ ID NO: 7) or NAVPNLRGDLQVLAQKVART (SEQ ID NO:8).

In a further embodiment, the alpha helical structure of the peptideenables the hydrophobic side chains of the residues LXXL/I to protrudefrom one side of the helix.

In a further embodiment, the alpha helical structure has at least oneturn.

In a further preferred embodiment, the peptide is between 7 to 45 aminoacids long, preferably between 7 and 40, 35, 30, 25, 20, or 15 aminoacids. For example, the peptide may be 7, 8, 9, 10, 11, 12, 13, 14, 22,24, 26, 28, 32, 34, 36, 38, 42 or 44 amino acids in length. In any case,the peptide of the present invention should not exceed a length whichwould allow the formation of tertiary structure, typically a peptideshould not exceed 45 amino acids if available as an isolated molecule.However, the peptide might exceed 45 amino acids if fused to a largermolecule such as an antibody or another protein or macromolecule whichcould prevent the formation of a tertiary structure within the peptide.Most preferably the peptide is 20 amino acids long.

In a further aspect, the peptides described herein may be linked to areadily detectable moiety. The term “readily detectable moiety” relatesto a moiety which, when located at the target site followingadministration of the peptides of the invention into a patient, may bedetected, typically non-invasively from outside the body and the site ofthe target located. Thus, the peptides of this embodiment of theinvention are useful in imaging and diagnosis. Readily detectable moietyare entities that are detectable by imaging techniques such as MagneticResonance Imaging (MRI), Magnetic Resonance Spectroscopy (MRS), SinglePhoton Emission Computed Tomography (SPECT) and Positron EmissionTomography (PET) and optical imaging. Preferably, imaging moieties arestable, non-toxic entities that retain their properties under in vitroand in vivo conditions. Examples of such moieties include but are notlimited to radioactive moieties, for example radioactive isotopes.Suitable radioactive atoms include technetium-99m or iodine-123 forscintigraphic studies. Other readily detectable moieties include, forexample, spin labels for MRI such as iodine-123 again, iodine-131,indium-111, fluorine-18, carbon-13, nitrogen-15, oxygen-17, gadolinium,manganese or iron and optical moieties which include Cy5.5 and quantumdots.

In a further embodiment of the present invention a polypeptide is linkedto a therapeutically active moiety, preferably the moiety is cytotoxic.

The term “therapeutically active moiety” encompasses a moiety havingbeneficial, prophylactic and/or therapeutic properties.

In one embodiment the therapeutically active moiety is a cytotoxicchemotherapeutic agent. Cytotoxic chemotherapeutic agents are well knownin the art and include anti-cancer agents such as:

Alkylating agents including nitrogen mustards such as mechlorethamine(HN2), cyclophosphamide, ifosfamide, melphalan (L-sarcolysin) andchlorambucil; 10 ethylenimines and methylmelamines such ashexamethylmelamine, thiotepa; alkyl sulphonates such as busulfan;nitrosoureas such as carmustine (BCNU), lomustine (CCNLJ), semustine(methyl-CCN-U) and streptozoein (streptozotocin); and triazenes such asdecarbazine (DTIC; dimethyltriazenoimidazolecarboxamide);Antimetabolites including folic acid analogues such as methotrexate(amethopterin); pyrimidine analogues such as fluorouracil(5-fluorouracil; 5-FU), floxuridine (fluorodeoxyuridine; FUdR) andcytarabine (cytosine arabinoside); and purine analogues and relatedinhibitors such as mercaptopurine (6-mercaptopurine; 6-MP), thioguanine(6-thioguanine; TG) and pentostatin (2′-deoxycofonnycin). NaturalProducts including vinca alkaloids such as vinblastine (VLB) andvincristine; epipodophyllotoxins such as etoposide and teniposide;antibiotics such as dactinomycin (actinomycin D), daunorabicin(daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin(mithramycin) and mitomycin (mitomycin Q; enzymes such asL-asparaginase; and biological response modifiers such as interferonalphenomes. Miscellaneous agents including platinum coordinationcomplexes such as cisplatin (cis-DDP) and carboplatin; anthracenedionesuch as mitoxantrone and antbracycline; substituted urea such ashydroxyurea; methyl hydrazine derivative such as procarbazine(N-methylhydrazine, MIH); and adrenocortical suppressant such asmitotane (o, p′-DDD) and aminoglutethimide; taxol andanalogues/derivatives; and hormone agonists/antagonists such asflutamide and tamoxifen.

Methods of conjugating polypeptides to therapeutic agents are well knownin the art.

In a further embodiment of the present invention a polypeptide is linkedto a particle that contains the therapeutic agent. Particles in thisinstance include nanoparticles and lipid-based vesicles such asliposomes or other similar structures composed of lipids. Accordingly,the present invention provides the peptides as defined herein and aliposome carrier and nanoparticles comprising the peptides as definedherein.

Liposomes are a spherical vesicles comprising a phospholipid bilayerthat may be used as agents to deliver materials such as drugs or geneticmaterial. Liposomes can be composed of naturally-derived phospholipidswith mixed lipid chains (egg phosphatidylethanolamine) or of purecomponents like DOPE (dioleolylphosphatidylethanolamine). The synthesisand use of liposomes is now well established in the art. Liposomes aregenerally created by sonication of phospholipids in a suitable mediumsuch as water. Low shear rates create multilamellar liposomes havingmulti-layered structures. Continued high-shear sonication tends to formsmaller unilamellar liposomes. Research has also been able to enableliposomes to avoid detection by the immune system, for examples bycoating the lipsomes with polyethylene glycol (PEG). It is also possibleto incorporate species in liposomes, such as the peptides of theinvention to help to target them to a delivery site, e.g. in cells or invivo.

The use of nanoparticles as delivery agents for materials associatedwith or bound to the nanoparticles is known in the art. Some types ofnanoparticle comprises a core, often of metal and/or semiconductoratoms, to which ligands of one or more different types may be linked,including, for example, one or more of the peptides of the presentinvention, see for example WO02/32404, WO2005/10816 and WO2005/116226.Other types of nanoparticle may be formed from materials such asliposomes. In some instances, the nanoparticles may be derivatised orconjugated to other ligands may be present to provide the nanoparticleswith different properties or functions. In some embodiments, thenanoparticles may be quantum dots, that is nanocrystals ofsemiconducting materials which have the striking chemical and physicalproperties that differ markedly from those of the bulk solid (seeGleiter, Adv. Mater. 1992, 4, 474-481). Now that their quantum sizeeffects are understood, fundamental and applied research on thesesystems has become increasingly popular. An interesting application isthe use of nanocrystals as luminescent labels for biological systems,see for example Brucher et al, Science 1998, 281, 2013-2016, Chan & Nie,Science, 1998, 281, 2016-2018, Mattousi et al, J. Am. Chem. Soc., 2000,122, 12142-12150, and A. P. Alivisatos, Pure Appl. Chem. 2000, 72, 3-9.The quantum dots have several advantages over conventional fluorescentdyes: quantum dots emit light at a variety of precise wavelengthsdepending on their size and have long luminescent lifetimes.

In a further embodiment, the cytotoxic moiety is a cytotoxic peptide orpolypeptide moiety by which we include any moiety which leads to celldeath.

Cytotoxic peptide and polypeptide moieties are well known in the art andinclude, for example, ricin, abrin, Pseudomonas exotoxin, tissue factorand the like. The use of ricin as a cytotoxic agent is described inBurrows & Thorpe, P.N.A.S. USA 90: 8996-9000, 1993, incorporated hereinby reference, and the use of tissue factor, which leads to localisedblood clotting and infarction of a tumour, has been described by Ran etal, Cancer Res. 58: 4646-4653, 1998 and Huang et al, Science 275: 25547-550, 1997. Tsai et al, Dis. Colon Rectum 38: 1067-1074, 1995describes the abrin A chain conjugated to a monoclonal antibody and isincorporated herein by reference. Other ribosome inactivating proteinsare described as cytotoxic agents in WO 96/06641. Pseudomonas exotoxinmay also be used as the cytotoxic polypeptide moiety (see, for example,Aiello et al, P.N.A.S. USA 92: 10457-10461, 1995.

Certain cytokines, such as TNFα and IL-2, may also be useful ascytotoxic and/or therapeutic agents.

Certain radioactive atoms may also be cytotoxic if delivered insufficient doses. Thus, the cytotoxic moiety may comprise a radioactiveatom which, in use, delivers a sufficient quantity of radioactivity tothe target site so as to be cytotoxic. Suitable radioactive atomsinclude phosphorus-32, iodine-125, iodine-131, indium-111, rhenium-186,rhenium-188 or yttrium-90, or any other isotope which emits enoughenergy to destroy neighbouring cells, organelles or nucleic acid.Preferably, the isotopes and density of radioactive atoms in thecompound of the invention are such that a dose of more than 4000 cGy,and more preferably at least 6000, 8000 or 10000 cGy, is delivered tothe target site and, preferably, to the cells at the target site andtheir organelles, particularly the nucleus.

The radioactive atom may be attached to the binding moiety in knownways. For example, EDTA or another chelating agent may be attached tothe binding moiety and used to attach ¹¹¹In or ⁹⁰Y. Tyrosine residuesmay be labelled with ¹²⁵I or ¹³¹I.

In a further embodiment, the present invention provides a polypeptide islinked to viral coat protein other than FMDV to change the trophism ofthe virus for delivery of DNA encoding therapeutic genes.

Alternatively, any of these systems can be incorporated into a prodrugsystem. Such prodrug systems are well known in the art.

In other aspects, the present invention use nucleic acid encoding apeptide as defined herein.

The term “nucleic acid coding for” (a peptide) relates to an RNA or DNAsequence which encodes a peptide comprising the sequence motif RGDLX⁵X⁶L(SEQ ID NO: 1) or RGDLX⁵X⁶I (SEQ ID NO: 2), wherein LX⁵X⁶L or LX⁵X⁶I iscontained within an alpha helical structure which can be used inaccordance with the invention or a functional variant thereof or aprecursor stage thereof, for example a propeptide or a prepropeptide.The peptide can be encoded by a full-length sequence or any part of thecoding sequence as long as the peptide is a functional variant. The term“variants” denotes all the DNA sequences which are complementary to aDNA sequence (reference sequence), which encode peptides used inaccordance with the invention, especially peptides as defined above ortheir functional variants and which exhibit at least approx. 70%, inparticular at least approx. 80%, especially at least approx. 90%,sequence identity with the reference sequence. The term “variants”furthermore denotes all the DNA sequences which are complementary to thereference sequence and which hybridize with the reference sequence understringent conditions and encode a peptide which exhibits essentially thesame activity as does the peptide encoded by the reference sequence, andalso their degenerate forms. It is known that small changes can bepresent in the sequence of the nucleic acids which can be used inaccordance with the invention; for example, without the property of afunctional variant being lost, these changes can be brought about by thedegeneracy of the genetic code or by non translated sequences which areappended at the 5′ end and/or the 3′ end of the nucleic acid. Thisinvention therefore also encompasses so-called “variants” of thepreviously described nucleic acids. The term “stringent hybridizationconditions” is to be understood, in particular, as meaning thoseconditions in which a hybridization takes place, for example, at 60° C.in 2.5×SSC buffer, followed by several washing steps at 37° C. in alower buffer concentration, and remains stable.

It is generally understood that the peptides and nucleic acids of thepresent invention can be of natural, recombinant or synthetic origin.Method of making, synthesising or modifying peptides are well known inthe art. Suitable methods include chemical synthesis, polymerase chainreaction (PCR) amplification, cloning or direct cleavage from a longerpolynucleotide. Polynucleotides of the invention have utility inproduction of the peptides of the invention, which may take place invitro, in vivo or ex vivo. The polynucleotides may be used astherapeutic or immunisation agents in their own right or may be involvedin recombinant peptide synthesis.

In a further aspect, the present invention provides a vector comprisinga nucleic acid as defined herein.

The vector of the present invention is preferably an expression vector,preferably a eukaryotic expression vector which may be adapted forpharmaceutical applications.

A “vector” refers to a structure consisting of or including a nucleicacid molecule which is suitable for transferring genetic material into acell. Typically a selected nucleic acid sequence is inserted into thenucleic acid molecule of the vector. Examples include plasmid and viralvectors. An “expression vector” is a vector constructed and adapted toallow expression of an inserted nucleic acid coding sequence in a cell.Thus, the vector includes nucleic acid sequences, which allow initiationof transcription in an appropriate location with respect to the codingsequence. Expression vectors can be adapted for expression inprokaryotic or eukaryotic cells, thus, a “eukaryotic expression vector”is constructed to allow expression of a coding sequence in a eukaryoticcell. Preferred examples of expression vectors of the present inventioninclude adenovirus, AAV and lentiviruses.

In a further aspect, the present invention provides a pharmaceuticalcomposition comprising peptide and/or nucleic acid and/or expressionvector as defined above and a pharmaceutical acceptable carrier.

The term “pharmaceutically acceptable carrier” generally includescomponents that are compatible with the peptide, nucleic acid or vectorand are not deleterious to the recipients thereof. Typically, thecarriers will be water or saline which will be sterile and pyrogen free;however, other acceptable carriers may be used. Typically thepharmaceutical compositions or formulations of the invention are forparenteral administration, more particularly for intravenousadministration.

In a further aspect, the present invention provides the use of a peptideand/or nucleic acid and/or expression vector according to the presentinvention for the preparation of a medicament for the treatment of aαvβ6 mediated disease or a disease where αvβ6 is overexpressed.

In a further aspect, the present invention provides a method of treatinga αvβ6 mediated disease comprising administering a peptide and/ornucleic acid and/or expression vector and/or pharmaceutical compositionas defined above to a patient. As mentioned herein, these conditionsmaybe in the general area of wound healing and inflammation.

Preferably, the disease is selected from chronic fibrosis, chronicobstructive pulmonary disease (COPD), lung emphysema, chronic woundingskin disease (e.g. epidermolysis bullosa) or cancer.

The medicament or pharmaceutical composition of the present invention asdefined above may usefully be administered to a patient who is alsoadministered other medicaments, as it will be known to those skilled inthe art. For example, in the case of cancer, the medicament orpharmaceutical composition of the present invention may be administeredto a patient before, after or during administration of the otheranti-tumour agent(s), for example before, after or during chemotherapy.Treatment with the peptide after chemotherapy may be particularly usefulin reducing or preventing recurrence of the tumour or metastasis. Forexample, the anti-tumour agent can be covalently linked directly orindirectly (via liposomes/nanoparticles) to a peptide of the invention.

In a further aspect, the present invention provides a method of imagingepithelial cells overexpressing αvβ6 in the body of an individual, themethod comprising administering to the individual an effective amount ofa peptide as defined above. The method is particularly useful for theimaging of chronic fibrosis, chronic obstructive pulmonary disease(COPD), lung emphysema, chronic wounding skin disease (e.g.epidermolysis bullosa) or epithelial tumour cells. For example, themethod of imaging may include linking the targeting peptide to afluorescent probe and incorporate into a mouth-wash, chewing gum, sprayor other emolument such that the αvβ6 bound peptide-probe conjugate maybe visualised by its fluorescent tag.

In a further aspect, the present invention provides a method for thediagnosis or prognosis in an individual of an αvβ6 mediated disease or adisease where αvβ6 is overexpressed, the method comprising administeringto the individual an effective amount of a peptide as defined above anddetecting the binding of the peptide to αvβ6.

In a further aspect, the present invention provides a method ofdelivering a therapeutic active moiety to a cell expressing αvβ6 or atissue containing cells expressing αvβ6 in a patient, the methodcomprising administering a peptide linked to a therapeutic active moietyas defined above to the patent.

In a further aspect, the present invention provides a method ofimproving the binding specificity of an αvβ6 binding peptide byincreasing or modifying the alpha helical content of the peptide. Forexample, the alpha helical content of the peptide may be increased bychanging the residues within sequence B_(n) RGDLXXLXXX Z_(m) (SEQ ID NO:5) into any other natural or synthetic amino-acid and measure the alphahelical content of the resultant peptides. Alternatively, peptide can beimproved by using the Saturation Transfer Difference NMR to determinewhich residues in a peptide are most likely to be interacting directlywith purified integrins (which may include α5β1, α8β1, αIIbβ3, αvβ1,αvβ3, αvβ5, αvβ6, αvβ8) and to subsequently insert residues asappropriate that possess particular side-chains, specific chargedistribution or other modifications that will decrease binding tonon-αvβ6 integrins or increase binding to αvβ6 integrins.

The term “improving the binding specificity” includes an increase in theaffinity of a peptide to αvβ6 compared to its affinity to anotherintegrin, for example αvβ3.

EXAMPLES

Cell Lines and Antibodies

Retroviral infection was utilised to generate αvβ6-positive and negativecell lines for this study. Mouse 3T3 fibroblasts and the human melanomacell lines A375P and DX3 were infected with retroviruses (Thomas et al;J Invest Dermatol.116(6):898-904, 2001) encoding human b6 and puromycinresistance gene to generate 3T3β6puro, A375Pβ6puro and DX3β6puro.Control cells expressed only puromycin (A375Ppuro and DX3puro-parental3T3 cells served as controls for 3T3β6puro, sometimes called 3Tβ6.19 orNIH3T3β6.19).

CHOβ6 cells, secreting recombinant-soluble αvβ6 lacking the cytoplasmicand trans-membrane domains of the integrin subunits. VB6 is a high αvβ6expressing oral squamous carcinoma (Thomas et al, 2001) and V(+)B2 is ahigh αvβ1-expressing human melanoma (Marshall et al 1995). A variety ofmouse monoclonal antibodies were used. Antibodies to αvβ3 (LM609),αvβ6(10D5) and a5 (P1D6) were purchased from Chemicon International,(emecula CA., USA). 63G9 (anti αvβ6) and 37E1 (anti-αvβ8). P2W7(anti-αv; produced in-house), L230 (anti-αv; from ATCC), P1F6(anti-αvβ5; a gift from Dr Dean Sheppard) and AIIB2 (anti-β1;purchasedfrom Developmental hybridoma), were produced in-house from theirrespective hybridomas. Fibronectin (F2006; Sigma Aldrich) wasbiotinylated using a kit (Amersham International, UK) according tomanufacturers instructions. All other reagents were purchased fromSigma-Aldrich unless stated otherwise.

Production of Recombinant Soluble αvβ6

CHOβ6 cells were grown to 80-90% confluency in RPMI supplemented with10% fetal calf serum (FCS), washed once with low serum medium (LowSM;RPMI 0.5% v/v FCS) and incubated for 48 hours in LowSM. Cell debris wasremoved from conditioned medium by centrifugation at 982 g and 0.1%(w/v) sodium azide was added as a preservative.

Conditioned medium was concentrated (up to 300-fold) and simultaneouslydiafiltrated against PBS using Centricon Plus-80 centrifugal filterdevices with a cut-off of 100 kDa (Millipore). The concentrate was addedto an immunoaffinity column that was generated by conjugating the mousemonoclonal anti-αv antibody L230 (in Coupling Buffer 0.1M SodiumPhosphate buffer, pH 7.0) to a 7 ml, gravity-flow agarose bead columnusing the Carbolink kit (Perbio Science UK Ltd) according to themanufacturers instructions. Recombinant soluble αvβ6 (rsαvβ6) was elutedwith 100 mM glycine pH 2.5-3.0 and neutralised immediately by additionof 300 μl 1 M Tris pH 7.5 to each 2 ml fraction. Peak fractions wereselected according to their absorbance at 280 nm and dialysed againstPBS using Amicon Ultra-15 centrifugal filter devices with a nominalmolecular weight cut-off (NMWCO) of 50 kDa (Millipore). Purity of theeluted protein was determined by SDS-PAGE and the concentrationdetermined by BioRad DC Protein Concentration Assay, using BSAstandards. Functional integrity of the rsαvβ6 was confirmed by showingthe integrin bound to fibronectin and latency-associated peptide(LAP)(αvβ6 ligands) immobilised to 96-well plates.

Cell Adhesion Assays

Adhesion of [⁵¹C/]-labelled cells to 96-well flexible plates coated withECM ligands has been described previously (Thomas et al, 2001). Briefly,plates were coated with LAP (0.25 μg/ml for NIH 3T3B6.19, 0.5 μg/ml forVB6) or vitronectin (10 μg/ml; BD Biosciences, Oxford, UK). Cells wereallowed to adhere for 40 minutes (VB6, NIH 3T3 β6.19) or 60 minutes(V+B2) before the plate was washed twice in PBS supplemented withcations (0.5 mM Mg²⁺, 1 mM Ca²⁺). Plates were cut with scissors and theradioactivity of each well quantified in a Wizard 1470 Automatic GammaCounter (Perkin-Elmer, Boston, Mass., USA). The percentage adhesion wascalculated by comparing the residual radioactivity associated with eachwell with the radioactivity of the initial input of cells as follows:

${{Adhesion}\mspace{14mu}(\%)} = {\frac{{Residual}\mspace{14mu}{radioactivity}\mspace{14mu}({cpm})\mspace{14mu}{of}\mspace{14mu}{well}}{{Radioactivity}\mspace{14mu}({cpm})\mspace{14mu}{of}\mspace{14mu}{input}} \times 100}$

All samples were tested in quadruplicate wells in at least threeseparate assays.

Competitive Sandwich ELISA

96-well plates (Immulon IB, Thermo LifeSciences) were coated with 10μg/ml P2W7 in PBS at 4° C. overnight then blocked by incubation with 2%(w/v) casein in PBS before washing with PBS. All subsequent washes usedWash Buffer (20 mM Tris, 150 mM NaCl, 1 mM MnCl₂), and all subsequentincubations took place in Conjugate Buffer (1% Casein, 20 mM Tris, 150mM NaCl, 1 mM MnCl₂). Wells were incubated with rsαvβ6 for one hour,washed, and exposed to a premixed solution of peptide and 2 μg/mlbiotinylated fibronectin. Bound biotinylated fibronectin was detectedwith ExtrAvidin HRP (SIGMA) at a dilution of 1:1000, and developed usingthe TMB+ system (DAKO). Assays were quantified by reading absorbance atA450 nm on a Tecan GENios plate-reader. All data was in the linearrange, confirmed by a standard curve of biotinylated fibronectin on eachplate.

Flow Cytometry

Expression of integrins by cell lines was assessed by flow cytometry asdescribed previously (Marshall et al., 1995). Briefly, cell suspensionswere incubated with anti-integrin antibodies at 10 μg/ml or biotinylatedpeptides at various concentrations. After 45′ at 4° C., cells werewashed and bound antibody/peptide was detected by 30′ incubation withmouse anti-biotin (1:100, Stratech, UK) followed by an additionalincubation for 30 minutes with Alexafluor 488 conjugated anti-mouse IgG(1:500 final dilution; Molecular Probes) or Streptavidin-FITC ((1:200final concentration) respectively. Cells were analysed on a FACScan(Becton-Dickinson) fitted with CellQuest software capturing 10,000events per sample.

Peptide Synthesis

Peptides were synthesised using standard solid-phase peptide synthesisby the Cancer Research UK Peptide Synthesis laboratory. Briefly,protected amino acids and preloaded Wang resins were obtained fromCalbiochem-Novabiochem (Nottingham, UK). Solvents and HBTU[2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate] were obtained from Applied Biosystems (Warrington,UK). The peptides were synthesised on a Model 431A updated and 433AApplied Biosystems Solid Phase Synthesiser on preloaded Wang resin usingbasic feedback monitoring cycles and HBTU as a coupling reagent.9-fluorenylmethyloxycarbonyl was used for temporary α-amino groupprotection and removed using piperidine. Side-chain protecting groupswere tert-butyloxycarbonyl for Lys; trityl for His, Asn and Gln;2,2,5,7,8-pentamethylchroman-6-sulphonyl for Arg; tert-butylester forGlu and Asp and tert-butyl ether for Thr, Ser and Tyr.

Cleavage from the resin and deprotection of the peptides was achieved bytreating the peptidyl-resin with 10 mls of a mixture containing 9.25 mlstrifluoroacetic acid, 0.25 mls ethanedithiol, 0.25 mlstriisopropylsilane and 0.25 mls water at 20° C. (room temp) for 4 hours.The peptide was precipitated using ice-cold diethylether and thenfiltered on a fine sintered glass filter funnel under light vacuum. Thepeptide precipitate was dissolved in 10% acetic acid/water solution andfreeze dried.

The crude peptides were purified by reverse phase HPLC on an AquaporeODS 20 micron 250×10 mm column and the authenticity of the purifiedpeptide was then confirmed by MALDI-TOF (matrix assisted laserdesorption ionization time of flight) mass spectroscopy on a FinniganMAT LCQ ion-trap mass spectrometer. Some peptides were biotinylated insitu on resin support using standard procedures.

NMR Sample Preparation

All NMR samples were prepared to a final volume of 300 μL for use in aShigemi BMS005V NMR tube by dissolving purified, freeze-dried peptide in2 mM phosphate buffered saline (PBS) at pH 6.4 with a phosphateconcentration of 25 mM and saline concentration of 100 mM. Forstructural studies, trifluoroethanol-d3 (TFE) was added as a helixstabilizer to provide a final concentration of 30% (v/v). SaturationTransfer Difference NMR (STDNMR) samples were prepared similarly withadditional components: 28 μM integrin αvβ6, 0.5 mM Mg²⁺ (added as MgCl₂)and 1.0mM Ca²⁺ (added as CaCl₂). STDNMR samples contained no TFE.

NMR Spectroscopy

All experiments were recorded on a Varian Unity INOVA 600 MHz NMRspectrometer with a z-shielded gradient triple resonance probe usingstandard procedures. Structural experiments, run at 10° C. for eachpeptide sample included two-dimensional (2D) nuclear Overhauser effectspectroscopy (NOESY), total correlation spectroscopy (TOCSY) androtating frame Overhauser effect spectroscopy (ROESY) experiments thatwere recorded with mixing times of 250, 70.0 and 100 ms respectively.These experiments were collected with 512 and 1024 complex points withacquisition times of 64 and 128 ms in the indirectly and directlyacquired 1H dimensions respectively. In addition, a two-dimensionaldouble-quantum-filtered correlated spectroscopy (DQFCOSY) experiment wascollected for each peptide at 10° C., with 1024 and 2048 complex pointswith acquisition times of 128 and 256 ms in the indirectly and directlyacquired 1H dimensions respectively. Slow exchanging amide protons weredetected from the fingerprint region of a 50 ms mixing time NOESYexperiment that was collected with 128 and 1024 complex points withacquisition times of 16 and 128 ms in the indirectly and directlyacquired 1H dimensions respectively. Data processing and analysis werecarried out on Sun Blade 100, Silicon Graphic Octane2 and Transtec X2100Linux workstations using NMRPipe (Delaglio et al, 1995) to process andNMRView (Johnson and Blevins, 1994) to view calculated structures.Saturation Transfer Difference NMR (STD NMR) experiments were run usingstandard saturation transfer experiment as described by Mayer and Meyer(1999, 2001), but incorporating a Hahn-echo filter as described by Yanet al, 2003. STD difference data were obtained at 25° C. with a spectralwidth of 6000 Hz, using a Hahn-Echo filter length of 30 ms and a totalnumber of data points and transients of 8192 and 16384 respectively. Onresonance irradiation was set to −2.5 ppm and off resonance irradiationwas set to −70.0 ppm. Irradiation was applied using a train of 9.4 msGaussian pulses, each with 100 Hz bandwidth with each pulse separated bya 1.7 ms delay. The total pulse train was applied for 2.0 s. In order toenable assignment of STD NMR transfer data, peptide assignments weremade from (2D) nuclear Overhauser effect spectroscopy (NOESY), totalcorrelation spectroscopy (TOCSY) and rotating frame Overhauser effectspectroscopy (ROESY) experiments obtained at 25° C. Resonance volumeintegrals were obtained using VNMR software operating on a SUN UNIXworkstation and the data analysed in accordance with the methodsoutlined by Mayer and Meyer (2001) to obtain the STD amplificationfactor using a ligand excess of 71.4. An individual amplification factorwas obtained for each amino acid residue from a sum of amplificationfactors from each 1H resonance for each residue. The residueamplification factor was converted to residue percentage STDamplification factors to enable a comparison with the highest residuefactor (that was given 100%).

Circular Dichroism

CD spectra were recorded on a Jasco J-600 spectropolarimeter at roomtemperature using 0.4 mM concentrations of peptide in buffers identicalto those used in the NMR investigations and containing TFE between 0-50%(v/v). Each solution was loaded onto 5 mm path length quartz cuvettesand each spectra obtained from an average of 4 scans at a range between190 and 260 nm, recorded at the speed of 20 nm/min, with a bandwidth of1 nm, a response of 2 s, and a resolution of 0.2 nm. The spectra areshown with no baseline correction. The OD values obtained by thespectropolarimeter were converted into ellipticity and adjusted to therelative peptide concentrations by the J-700 windows standard analysis(v.1.50.01) software. The ellipticity values at 3 wavelengths: 222, 208and 192 nm were then converted into the mean ellipticity (meanq)obtained for each peptide at TFE concentrations between 0-50%(v/v) usingthe approach as described in Forood et al. 1993.

Structural Calculations and Analysis

All structural calculations were obtained using the Crystalography andNMR System (CNS) version 1.1 running on Silicon Graphics Octane2 andTranstec X2100 Linux workstations (Brunger et al, 1998). All NOE and ROEcontacts were classified into one wide classification between 2.5-5.0 Åwith final structures calculated from extended coordinates using thestandard CNS NMR anneal protocol with sum averaging for dynamicannealing with constraints from both extended and folded precursors. Afinal structural ensemble of 40 structures for each peptide was producedwith all structures used to produce statistical energy and root meansquare (r.m.s.) deviation structural information. Backbone and heavyatom r.m.s. deviation values were obtained using MOLMOL version 2k.2(Koradi et al, 1996) on a PC running Microsoft Windows 2000. Thestructural integrity of each ensemble was evaluated using PROCHECK-NMR(Laskowski et al, 1996) run on a Transtec X2100 Linux workstation.Energy comparisons between structure ensembles created in CNS were madeusing GROMOS96 43B1 parameter set (van Gunsteren, 1994) within DEEPVIEWversion 3.7 (Guex and Peitsch, 1997).

Results

Peptides Derived from αvβ6 Ligands Confirms Requirement for DLXXL

The integrin αvβ6 binds to its ligands, in part, through recognition ofthe peptide motif RGD (Arg-Gly-Asp). Ligands of the integrin αvβ6include the TGFβ latency associated peptide (LAP), which we have foundto be a highly specific αvβ6 adhesive ligand, fibronectin and certainviruses including foot-and-mouth-disease virus (FMDV). We thereforechose to examine overlapping 7-12 mer linear peptides from known αvβ6ligands, that included the necessary RGD motif, from LAP and twoserotypes of FMDV (DD1-DD19). In initial studies, the peptides weretested at a concentration of 500 μM for their ability to inhibitαvβ6-dependent adhesion to LAP of the tumour cells 3T3β6.19 and VB6. Itwas shown that the most potent peptides had the sequence DLXXL (or thesimilar DLXXI), a sequence whose importance had been discoveredpreviously (Kraft et al, J Biol Chem. Jan. 22; 274(4):1979-85. 1999).

Second Generation αvβ6-Targeting Peptides: Ligand-Based, 20 mer,RGDLXXL/I SEQ ID NO: 1/SEQ ID NO: 2) Peptides

There was also a suggestion that longer peptides or at least those withmore residues C-terminal to the RGD motif, were more potent inhibitorsof αvβ6-dependent adhesion, compared to peptides with additionalN-terminal sequence. To examine this possibility, we generated 20 merpeptides with extended C-terminal regions derived from LAP β1(A20LAP)and the foot and mouth disease virus serotypes C-S8c1 (A20 FMDV1—Mateuet al.,1996) and O1BFS (A20 FMDV2—Logan et al., 1993) and repeated theexperiments.

A20 LAP GFTTGRRGDLATIHGMNRPF (SEQ ID NO: 6) A20 FMDV-1YTASARGDLAHLTTTHARHL (SEQ ID NO: 7) A20 FMDV-2 NAVPNLRGDLQVLAQKVART(SEQ ID NO: 8)

FIG. 2 confirms that A20 LAP, A20 FMDV and A20 FMDV2 were far morepotent at inhibiting αvβ6-dependent binding of 3T3β6.19 (FIG. 2A) andVB6 (FIG. 2B) to LAP. Thus whereas the IC50 of DD1 inhibition of3T3β6.19, the best short LAP peptide, is 216 μM (data not shown), theIC50 for A20 LAP is 13.8 μM. Similarly, the IC50 for DD19, the shortFMDV2 peptide, is 190 μM compared with 1.2 μM for A20FMDV2.

20 mer RGDLXXL/I (SEQ ID NO: 1/SEQ ID NO: 2) Peptides are more PotentInhibitors of αvβ6-Dependent Cell Adhesion than Shorter RGDLXXL/I (SEQID NO: 1/SEQ ID NO: 2) Peptides

In order to test the hypothesis that an extended C-terminal sequenceincreases the efficacy of anti-αvβ6 peptides, the αvβ6-specific activityof A20LAP was compared with shorter versions of the same peptide, DD1, 2and 3. A20LAP was significantly better at inhibiting αvβ6-dependent celladhesion of 3T3β6.19 to LAP-coated plates. Thus, the number of aminoacids C-terminal to RGD for A20LAP, DD1 and DD3 is 11, 5 and 4,respectively. This replicates the potency order of the peptides; thus inthe presence of 20 μM A20-LAP, DD1 and DD3, adhesion of 3T3β6.19 to LAPwas just 32±7%, 57±4% and 79±22% of control cell adhesion respectively.In addition, the experiments were repeated using another cell line, VB6.VB6 is a human oral squamous cell carcinoma that expresses high levelsof αvβ6 (Thomas et al, 2001b). It is thus a more appropriate model sinceentirely human αvβ6 is expressed in its natural, epithelial environment.Similarly to 3T3β6.19, adhesion of VB6 to LAP is abrogated by theαvβ6-blocking antibody 63G9 and is, therefore, considered entirelyαvβ6-dependent. Whilst difficult to quantitate due to intra-assayvariation, the same pattern as was seen with 3T3β6.19 is broadlyobservable in assays using VB6. Thus at 100 μM DD1, DD2 and DD3 havelittle effect on VB6 adhesion to LAP, while the longer peptide A20LAPcompletely blocks cell adhesion at this concentration.

Similarly, A20FMDV2 is a markedly better inhibitor of VB6 adhesion toLAP than DD19, a shorter peptide based on the same amino acid sequence.The effect here is more dramatic: cell adhesion in the presence of 20 μMDD19 is the same as adhesion in the absence of peptide, however adhesionis reduced to background levels in the presence of 20 μM A20FMDV2.

Competitive Sandwich ELISA

To see if this pattern was repeatable in an isolated protein assay,competitive sandwich ELISAs were performed. Briefly, 96-well plates werecoated with the anti-αv antibody P2W7 by incubating overnight at 4° C.Remaining non-specific binding sites were blocked by incubation with asolution of 2% (w/v) casein in PBS. Wells were then incubated withrsαvβ6 before washing and exposure to a pre-mixed solution ofbiotinylated fibronectin and peptide. Bound biotinylated fibronectin wasdetected with peroxidase-conjugated ExtrAvidin. Nine-point dose responsecurves were generated using seven concentrations of peptide and positiveand negative controls, and an IC50 concentration determined using asigmoidal curve-fit model with GraphPad Prism software.

Interestingly, the pattern seen in competitive sandwich ELISA isslightly different to that seen in cell adhesion assays. Although theshort LAP-based peptides DD2 and DD3 have a significantly lower IC50than the longer peptide A20LAP, the short peptide DD1 has a very similarIC50 to A20LAP.

TABLE 5 Mean Standard Sequence IC50 Deviation  Peptide (SEQ ID NO) (nM)(nM) DD1      RRGDLATIH (9) 9.2 0.7 DD2  FTTGRRGDLATI (10) 30.9 3.3 DD3   TGRRGDLATI (11) 22.6 4.0 A20LAP GFTTGRRGDLATIHGMNRPF (6) 6.7 0.9 DD19  VPNLRGDLQVLA (12) 85.0 31.1 A20FMDV2 NAVPNLRGDLQVLAQKVART (8) 15.6 5.3

Analysis of 20 mer RGDLXXL/I (SEQ ID NO: 1/SEQ ID NO: 2) Peptides byCell Adhesion Assay

The three 20 mer peptides A20LAP, A20FMDV1 and A20FMDV2 were assessedfor inhibition of αvβ6-dependent cell adhesion. Multiple concentrationsof peptide were used in order to generate inhibition curves, from whichIC50 values were calculated using Prism Software as shown in the tablebelow. In both 3T3β6.19 and VB6 assays, A20FMDV2 was the most potentinhibitor of αvβ6-dependent cell adhesion, followed by A20LAP. A20FMDV1was the least potent inhibitor in both assays. Therefore, predictedhelicity correlates with peptide potency in inhibition of αvβ6-dependentcell adhesion assays. Interestingly, the IC50s for all peptides wereapproximately 1,000-fold higher in cell adhesion assays than incompetitive ELISAs; this effect has been reported previously foranti-αvβ3 peptides (Goodman et al, 2002).

TABLE 6 Peptide Sequence 3T3β6.19 VB6 A20FMDV1 YTASARGDLAHLTTTHARHL86.5 ± 49.9 μM 38.2 ± 31.1 μM (SEQ ID NO: 7) A20LAP GFTTGRRGDLATIHGMNRPF13.8 ± 3.3 μM 28.7 ± 11 μM (SEQ ID NO: 6) A20FMDV2 NAVPNLRGDLQVLAQKVART 1.2 ± 0.2 μM 1.54 ± 0.4 μM (SEQ ID NO: 8)

Binding Hierarchy of 20 mer Peptide Antagonists

To compare the binding abilities of each peptide for αvβ6, rsαvβ6 wasimmobilised on 96-well plates. Various concentrations ofbiotinylated-A20 FMDV1, -A20 LAP or -A20 FMDV2 were added to the platesfor 45′ in the presence of 1 mM Ca²⁺ and 0.5 mM Mg²⁺ ions. Bound peptidewas detected with streptavidin-HRP. In addition, biotinylated, scrambledversions of each peptide were also tested. FIG. 3 shows that the abilityto bind αvβ6 followed the order A20 FMDV2, A20 LAP and A20 FMDV1. Thusat all concentrations A20 FMDV2 bound more strongly to the immobilisedrsαvβ6. At 10 nM concentrations each peptide still exhibited nearmaximal binding in contrast to scrambled controls which showed nobinding. Even at 1 nM A20 FMDV2 showed 50% maximal binding.Interestingly, scrambled A20 LAP did show binding at 10 μM and 1 μM incontrast to scrambled FMDV peptides which showed little binding at anyconcentration tested.

AGADIR Predicts Helical Propensity of 20 mer αvβ6 Antagonists

For both LAP- and FMDV-based peptides, longer peptides inhibited αvβ6with a greater degree of potency than shorter peptides, even when allthe peptides concerned contained the crucial RGDLXXL/I (SEQ ID NO: 1/SEQID NO: 2) motif. However, aside from this motif, there are no obvioussimilarities between the sequences of A20LAP and A20FMDV2; therefore itwas considered unlikely that the motif had simply been extended in thelonger peptides. Another possible explanation was that the increasedlength causes a change in the affinity of the peptide for αvβ6 bychanging the presentation of the RGDLXXL/I (SEQ ID NO: 1/SEQ ID NO: 2)motif. The presence of secondary structure, which could potentiallystabilise active conformations of RGDLXXL/I (SEQ ID NO: 1/SEQ ID NO: 2),was therefore considered.

Intuitively, long linear peptides are more likely to be able to assumemany more shapes in three-dimensional (3D) space than shorter peptidesand few of these long shapes are likely to be optimal ligands for areceptor. Since in our experiments the longer peptides were more potentinhibitors of αvβ6 than shorter peptides this suggested that there maybe secondary structure minimising the number of possible 3Dconformations. In solution, the VP1 coat protein is unstructured (Loganet al 1993). However, Logan and colleagues characterised the crystalstructure of the immunogenic G-H loop of the VP1 domain of FMDV. Theyreported that the loop appeared to have a helical structure in thecrystal. Thus we considered that our 20 mer peptides may also havehelical structures which thermodynamically would stabilize thestructure. We inserted the amino-acid sequences into AGADIR (Munoz andSerrano, 1994, 1995) software that predicts helical propensity withinpeptides. The software assigns probability values that individualresidues in a peptide sequence are part of a helical structure. Thissoftware is widely acknowledged as a reasonably accurate method ofpredicting helicity. FIG. 3 shows that all three 20 mer peptides arepredicted to have a helical propensity in the DLXXL/I region in theorder A20 FMDV2>A20 LAP>A20 FMDV1 but that A20 FMDV2 has a much greaterpredicted helical propensity than either A20 LAP or A20 FMDV1 and itextends beyond DLXXL/I. Thus, the predicted helical propensity of the 20mer peptides correlates with their potency as αvβ6 antagonists. To testthis hypothesis the three 20 mer peptides were studied in more detail,both biochemically and structurally.

Far-UV CD Analysis

Circular dichroism is an optical technique based on the changes inpolarisation that occur when UV light passes through a chiralenvironment. Differential absorption of left- and right-polarised lightcauses circularly polarised light to become elliptically polarised. Thediffering chiral environments of the different forms of secondarystructure (beta-sheets, turns and helices; also the unstructured ‘randomcoil’ state) cause each to have their own characteristic far-UV CDspectra; therefore CD can be used to study the amount of each type ofsecondary structure in a particular protein or peptide.

To confirm whether our 20 mer peptides formed helical structures wedetermined the far-ultraviolet circular dichroism (Far-UV CD) spectrafor each peptide in increasing concentrations of the helix stabilizer,TFE (FIG. 1). To enable cross-comparison between samples, the meanresidue ellipicity (θ-[q]222) for each peptide in TFE proportionsbetween 0-50% (v/v) in PBS are shown in FIG. 1. FIGS. 2( a-c) eachillustrates an isodichroic point at 202 nm that indicates that atwo-state equilibrium exists between the unfolded and the helicalpeptide state for each peptide (Khandelwal et al, 1999). The meanmolecular ellipticity identifies that both A20FMDV2 and A20LAP undergoestransition to helix between 10-25% TFE whereas A20FMDV1 undergoestransition over a much wider concentration range (10-40% v/v in PBS) ofTFE. Thus, CD data show that if a stabilizing influence is present allthree 20 mer peptides form helices in their structure but that there isan increased propensity for A20 FMDV2 and A20 LAP to form helicescompared with A20 FMDV1. Empirically-determined helical propensitytherefore correlates with anti-αvβ6 potency.

NMR Analysis of αvβ6 20 mer-Peptide Antagonists

Spin systems were identified by analysis of two-dimensional DQF-COSY andTOCSY NMR spectra together with resonance assignments and all theobserved ¹H chemical shifts are listed in Table 3. Assignments for themajority of nuclei in all ¹H spin systems were possible for A20FMDV1,A20FMDV2 and A20LAP peptides except for Thr2 of A20FMDV and Gly1 ofA20LAP.

Through-space assignments were achieved using two-dimensional NOESY andROESY spectra of each peptide in 30% TFE (v/v). Amides in slow exchangeand deemed capable of being hydrogen bond donors were originallyidentified from a NOESY experiment obtained after re-suspension of eachpeptide in D20 and confirmed by visual inspection of intermediatestructures from CNS calculations. Additional f restraints were obtainedfrom application of the Karplus relationship to 3JHNHa that wereobtained from high-resolution DQF-COSY spectra. 3JHNHa values less than5 Hz were used to constraint f for that residue to −60°±30°. A cut offvalue of 5 Hz was used to allow for the fact that 3JHNHa values obtainedby DQF-COSY are always larger than those obtained by more accurateheteronuclear NMR methods (Cavanagh et al., 1996).

The contact distribution of NOE and ROE was found to be greater forresidues in the C-termini following from RGD in each of the peptidesstudied. A summary of the number of contact types and additionalrestraints are shown in Table 4 with the distribution of restraintsacross each peptide shown in FIGS. 3( a), (b) and (c). Contact typesobserved in FIGS. 3( a) support standard helix conformations directlyC-terminal to the RGD motif with contacts observed between Ha:i andHN:i+3 as well as Ha:i and Hb:i+3. Additionally, slow HN exchange and3JHNHa values less that 5 Hz were observed in some residues as shown inFIG. 6.

FIG. 4 highlights the main helical contact regions of 2D NOESY spectrafor all three peptides and demonstrates that the number of contacts andresonance dispersion is greatest with A20FMDV2 and least with A20FMDV1.Contacts that support helical character appear most sporadic in A20FMDV1and best defined in A20FMDV2 with the A20LAP contact distributionfalling between these two extremes.

Structure Calculations and Analysis

NMR was used to determine the solution structures of the three 20 merpeptides, and thus to confirm the CD and the in silico (AGADIR) data.NMR data generates a series of constraints, for example in the form ofNuclear Overhauser Effects (NOEs). NOEs are observed when two atoms areclose enough in space for NMR spectroscopic relaxation to occur betweenthem. If the two atoms are identified as being non-adjacent in theprimary sequence, each NOE provides evidence to support the presence ofsecondary structure that maintains these regions in close proximity.Furthermore, the NOEs provide distance constraints that can be tabulatedand used in tandem to produce a model of the structure. Constraints suchas these limit the number of peptide conformations that are physicallypossible; a computer algorithm is then used to generate a number ofconformations (known as ensembles) that fit the constraints.

All structural data was determined using CNS as described in theexperimental procedures. No calculated structure gave violations greaterthan 0.2 Å or bond angle violations greater than 5° when all 40structures were used to compute the ensemble average structural set.Structural energy statistics and backbone r.m.s. deviations for allthree peptides are all shown in Table 3 and all ensembles and ensembleaverage structures are shown in FIG. 5. Backbone r.m.s. deviations arequoted over residues LXX[L/I]XX for each peptide to enable comparativeanalysis of each peptide. PROCHECK-NMR analysis for each of the40-structure ensembles identified that 94.3, 94.8 and 93.6% of allresidues fell in the allowed regions of the Ramachandaran plot forA20FMDV1, A20FMDV2 and A20LAP respectively. Residues that fell outsidethe allowed regions were from the first four amino acids in eachensemble and their deviations were consistent with data obtained fromstructure calculations for regions where little or no restraint data isgiven. Helix limits shown in FIG. 3 and FIG. 5 were identified from thedihedral angle and hydrogen bond geometry obtained from the calculatedstructural ensembles and not from the original data. This approachenabled the combination of all structural information to contribute tothe geometrical characteristics of each peptide. The helix associatedresidues for each peptide were identified as Ala10-Thr14 for A20FMDV1;Leu10-Val17 for A20FMDV2 and Leu10-Gly15 for A20LAP.

40 structure ensembles for A20FMDV1, A20LAP and A20FMDV2 are shown inFIG. 5. All three peptides show a similar structure, with the RGDsequence forming the head of a loop which is followed immediately by ahelical region. The Arginine and Aspartate residues point outwards fromthe loop, forming a kind of hammerhead similar to that observed in thecrystal structure of αvβ3 bound to an RGD peptide (Xiong et al, 2002).The helical region varies in length between the three peptides. A20FMDV2has the greatest degree of ordered structure (FIGS. 5E & F) and thelongest helix, containing approximately three turns. A20LAP has aslightly shorter helix and A20FMDV1 has a very short helix, consistingof only one turn. Thus helical structure in the LXXL/I region correlateswith biological anti-αvβ6 activity.

Thus, NMR analysis confirmed CD data that all three peptides had helicesin their structure and that the location of this extended α-helicalelement was directly C-terminal to the RGD motif. Residue i-j contacts,as shown for NH-NH in FIG. 3, identifies constraints that make all threepeptides adopt a turn conformation that enables the RGD motif to bepresented at the turn of a hairpin structure. Long-range contacts wereobserved between Ala3-Thr17 and Ser4-Thr15 for A20FMDV; Val3-Arg19,Val3-Thr20, Pro4-Val17, Leu6-Val12 and Gly8-Val12 for A20FMDV-2 andPro2-Ala11, Pro2-Ile13, Pro2-His14, Thr4-Ala11 and Gly5-His14 forA20LAP.

Saturation Transfer Difference MIR

It is clear that the length of the α-helices C-terminal to the RGD motifin the three 20 mer peptides, increases with increasing efficacy of thepeptides (see FIG. 3). These data suggest that the length or stabilityof the helix may contribute to the potency of the peptides to functionas αvβ6 antagonists. However, the NMR identification of an α-helixC-terminal to RGD in our peptides was performed in the presence of ahelix stabilizing solvent, TFE. To determine whether the peptides are inthe form of a helix when associated with αvβ6 in physiological buffer weutilized saturation transfer difference NMR. STD NMR spectra identifyingthe interactions of the most potent αvβ6 antagonist peptide, A20FMDV2,with the integrin αvβ6 are shown in FIG. 6. Analysis of the STDdifference spectrum was made possible by the reasonable dispersion ofNMR resonances in this peptide in the absence of TFE. Where degeneratechemical shifts created overlap, any STD difference values wereattributed equally to both nuclei in order to remove any potential biasfrom the data. Control 1H STDNMR spectra FIG. 6( a) and FIG. 6( c)highlight all 1H NMR resonances from the peptide and STD differencespectra shown in FIGS. 6( b) and 6(d) highlight those 1H resonances thathave been in proximity to the integrin during the binding event. FIGS.6( c) and 6(d) enable the identification of key contact points includingHd and Hb of Leu13 and Leu10 as well as Arg7 Hb/Hd, Thr20 Hg and Lys16Hb/Hd. Key resonances illustrating reduced or absent STD differencespectra included Leu6 Hd and Hg of Gln11, Val12 and Gln15 and Val17. STDamplification factors for individual nuclei were calculated from thisdata to be from 0.0 to 8.81 with residue sum amplification factorsobserved for all residues in A20FMDV2. The relative STD amplificationfactor across all residues of A20FMDV2 are shown in FIG. 7 andidentifies that contact is highlighted across the entire peptide withmajor interactions observed for Arg7, Asp9, Leu10, Val12, Leu13, Lys16,Val17 and Thr20. These data may suggest that contacts beyond the DLXXL/Ihelical motif are important for improved binding to αvβ6.

Presence of Helical Structure in αvβ6-Bound Peptide

The presence of the helix causes the non-consecutive leucine andleucine/isoleucine residues of the DLXXL/I motif to be brought intojuxtaposition, thus forming a small hydrophobic patch. Sinceinteractions between hydrophobic patches are one of the classicmechanisms for protein-protein binding, it is possible to hypothesisethat the Leucine-Leucine or Leucine-Isoleucine patch brought about bythe helix is involved directly in binding of the peptides to αvβ6. Thiswould explain why the identity of the ‘XX’ residues is less importantthan the leucine and leucine/isoleucine residues in the DLXXL/I motif(Kraft et al, 1999). In order to test this hypothesis we employedSaturation Transfer Difference (STD) NMR, a technique which measuresenergy transfer from a large protein, in this case rsαvβ6, to a muchsmaller molecule, in this case the peptide A20FMDV2. The technique workson an atom-specific basis and gives a measurement of proximity ofindividual residues in a small ligand (A20FMDV2) to a large, receptorprotein (rsαvβ6). In this way it is possible to gain an indication ofthe precise residues involved in binding of the peptide to the receptor.Excluding Arg⁷, which as part of the RGD motif is expected to exhibitstrong contacts with αvβ6, the residues with the highest levels ofenergy transfer are Leu¹⁰, Leu¹³, Lys¹⁶ and Val¹⁷; thus major contactswith αvβ6 have a regular periodicity of approximately three residues.This is strongly indicative of the presence of helical structure onbinding. It is important to note that, unlike the solution NMRexperiments, the STD NMR was carried out in physiological buffer (PBS)and in the absence of the helix-stabilising alcohol TFE. Therefore thisis strong evidence that although A20FMDV2 exists in solution inequilibrium between helical and random-coil states, the αvβ6-boundpeptide exists in a predominantly helical state. Indeed, when theresidues that show the highest degree of close contact with αvβ6 aremapped onto the mean 3-dimensional structure of A20FMDV2 in 30% TFE,these residues align on a single face of the peptide. This stronglyindicates that the presence of helical structure brings these otherwisenon-adjacent residues into juxtaposition, forming a single binding-facefor direct interaction with αvβ6.

An α-Helix is Required for Optimal Binding to αvβ6

The data above shows clearly that when A20FMDV2 binds to αvβ6 there isan α-helix C-terminal to RGD. Moreover, by bringing into juxtapositionthe two non-contiguous leucines at L10 and L13 this allows for a closecontact between the ligand (A20 FMDV2) and the integrin. In order toprove that the α-helix was required for ligand binding to αvβ6, wesynthesized three A20 FMDV2 variant peptides that replaced L-valineswith D-valines at positions D12 and D17. FIG. 4 shows that each of thesevalines is predicted to be within the α-helix formed by A20FMDV2, whichwas confirmed by NMR. By inserting D-valines, we would expect to disruptthe helical nature of the peptide without removing the possibility ofkey contact residues (Arg7, Asp9, Leu10 and Leu13) from interacting,while maintaining other aspects of the peptide, such as chargedistribution and pH.

The D-Valine peptides were analysed in cell adhesion assays with3T3β6.19 and VB6 cell lines and the data summarised in the table below.The results indicate that the L-to-D changes have a cumulative effect:while peptides DV12 and DV17 have IC50s approximately three times higherthan that of ‘parent’ peptide A20FMDV2, the efficacy of DV1217 isreduced by approximately 20-fold in VB6 assays and 40-fold in the3T3β6.19 assays, see the table below.

TABLE 7 IC50 values for D-Valine-containing peptides in cell adhesionassays. 3T3β6.19 VB6 Peptide IC50 (μM) SD (μM) n IC50 (μM) SD (μM) nA20FMDV2 1.2 0.2 4 0.96 0.16 3 DV12 ND ND 0 3.35 0.65 3 DV17 ND ND 02.97 2.02 3 DV1217 48.5  37 4 22.81 N/A 2 SD, standard deviation; n,number of experiments; ND, not determined.

Peptide DV1217 was also compared to A20FMDV1, A20LAP and A20FMDV2 in anisolated receptor binding assay, using peptides synthesised with anN-terminal biotin. Briefly, 96-well plates were coated with rsαvβ6 andremaining non-specific protein binding sites blocked by incubation with1% (w/v) casein in PBS. Wells were incubated with biotinylated peptidesbefore washing and subsequent detection of bound peptide with ExtrAvidinHRP. Biotinylated peptides bound specifically to immobilised rsαvβ6, asthere was no binding in the absence of rsαvβ6. Binding wassequence-specific, as control peptides with scrambled sequences boundvery little in comparison with the original sequences, and showed nobinding at all at concentrations below 100 nM. Peptide A20FMDV2 showed ahigher degree of binding to αvβ6 than A20LAP, and both bound more thanA20FMDV1. Peptide DV1217, which except for the isomerism of D-Val¹² andD-Val¹⁷ and consequent lack of helical structure is chemically identicalto A20FMDV2, only bound as well as A20FMDV1. Thus, helical structurecorrelates with binding to rsαvβ6 in isolated protein assays as well asin inhibition of cell adhesion assays. These data also show that whilethe presence of helical structure promotes binding to αvβ6, thepotential to form helical structure is not a pre-requisite for binding;as evidenced by the dose-dependent binding of A20 DV1217.

To confirm that the D-valine substitutions had in fact disrupted helixformation we analysed the double-mutant by CD and NMR. The CD data showthat the DV1217 mutant was unable to form a helix even in 50% TFE andthe NMR analysis that helix formation was not predicted from 40overlapping ensembles. Since there were only structural differences, nosequence or charge differences between A20 FMDV2 and the DV1217 doublemutant, these data suggests strongly that an α-helix C-terminal to RGDis an essential component of an optimal αvβ6-specific binding motif.

P18-INK6 Derived Peptides

Whereas A20FMDV1, A20FMDV2 and A20LAP peptides were derived from proteinsequences that are known to bind integrin αvβ6, we investigated whetherother sequences that contain the RGDLXXL/I (SEQ ID NO: 1/SEQ ID NO: 2)sequence motif whereby the LXXL/I motif is contained within analpha-helical structure. We chose the motif contained in the P18-INK6gene (also known as Cyclin-dependant kinase 4 inhibitor C or P18-INK4c)with sequence shown below.

(SEQ ID NO: 12) DD19 VPNLRGDLQVLA (SEQ ID NO: 13) P18-INKSAAARGDLEQLTSLLQNNVNV

The P18-INK sequence contains the RGDLXXL (SEQ ID NO: 1) sequence andwhen analysed using the AGADIR software and showed that the LEQLsequence in P18-INK peptide formed an alpha-helical motif. This sequencewould therefore be predicted to have αvβ6-binding properties, despitethe limited likelihood of this being a physiological interaction becausethe αvβ6 ligand-binding site is extracellular while p18-INK6 isintracellular.

Comparison of the binding affinity of the P18-INK with that of DD19 (aRGDLXXL (SEQ ID NO: 1) peptide with LXXL sequence not in an alphahelical structure) to integrin αvβ6 showed that the binding affinity ofP18-INK was significantly greater than that of DD19 (FIG. 8). Thisindicates that RGDLXXL (SEQ ID NO: 1) sequences which are contained inproteins not known to bind αvβ6 but which contain the LXXL motif as partof an alpha-helix still bind αvβ6 when presented in isolation.

In Silico Modelling of P-INK Peptides Using AGADIR

In addition, it was decided to use this system to explore thepossibility of using in silico design (via the AGADIR algorithm) toenhance peptide helicity, and thereby potentially enhance anti-αvβ6potency. Different ways of combining the A20FMDV2 and p18-INK sequenceswere looked at, and the one which gave the highest degree of predictedhelicity in the LXXLXX region (INK-FMDV) was chosen for further study.Subsequently, two more peptides were made, with single amino acidchanges: the first, INK-FMDV-X, increased the overall predicted helicityof the peptide; the second, pINK-FMDV2-XX, increased the predictedhelicity of the LXXLXX region while decreasing the predicted helicity ofthe RGD.

These peptides were analysed using the Screening ELISA. Briefly, rsαvβ6was immobilised on the surface of 96-well plates by exposure to platescoated with an anti-αv monoclonal antibody (P2W7). The immobilisedrsαvβ6 was then exposed to a mixture of peptide andbiotinylated-fibronectin for one hour, after which unbound material waswashed away and bound biotinylated-fibronectin detected withExtrAvidin-HRP. Serial dilutions of peptide allowed the generation of adose-response curve, from which an IC50 was calculated using a sigmoidalcurve-fit model (Prism software).

The results showed that a 20 mer peptide derived from the p18-INK6sequence is a functional inhibitor of recombinant αvβ6, with an IC50 of23 nM in competitive ELISA. The peptide P-INK also inhibitedαvβ6-dependent adhesion in a preliminary cell adhesion assay.

Peptides derived from the intracellular protein p18-INK6 are thereforecapable of inhibiting recombinant and cellular αvβ6. This is unlikely tohave a physiological impact as the ligand-binding domain of αvβ6 isextracellular and therefore unlikely ever to ‘see’ p18-INK6; howeverthese data lend support to the model proposed here, that an RGDLXXL (SEQID NO: 1) motif with a helical tendency in the LXXL region is likely topossess αvβ6-binding activity.

Assessment of Peptide Specificity by Flow Cytometry

Biotinylated peptides were allowed to bind to A375Ppuro and A375Pβ6puroand binding detected with a mouse anti-biotin antibody followed byAlexaFluor488-conjugated goat anti-mouse. The use of a secondaryantibody that bound biotin provided an important amplification step, aspreliminary experiments using direct detection with streptavidin-FITCresulted in little or no detectable signal. The peptides were tested atseveral different concentrations and demonstrated concentration-specificdifferential binding to the A375Pβ6puro cell line. DV1217 was highlyspecific for A375Pβ6puro, as it did not bind noticeably to A375Ppuro atany of the concentrations tested (up to 100 μM), but bound to A375Pβ6 at10 μM and at 1 μM. A20FMDV2 did bind to A375Ppuro, but only at 10 μM,whereas binding to A375Pβ6puro was observed at 10 μM, 1μM, 0.1 μM, 0.01μM and 0.00 μM; differential binding of four orders of magnitude.A20FMDV1 was also relatively specific, showing binding to A375Pβ6puro at1 μM, a concentration at which it did not bind to A375Ppuro. A20LAPshowed relatively little specificity for A375Pβ6puro and bound to bothcell lines at 10 μM and 1 μM, although binding to A375Pβ6puro wasslightly greater at both concentrations.

All the peptides contained an RGDLXXL/I (SEQ ID NO: 1/SEQ ID NO: 2)motif; therefore the presence of this motif is not a guarantee ofspecificity for αvβ6. In addition, of the four peptides tested, the twopeptides with the most stable (A20FMDV2) and the least stable(A20DV1217) helices in the post-RGD sequence were the most specific forαvβ6 over the other RGD-directed integrins present; therefore helicityin the post-RGD region does not provide specificity for αvβ6. However,these data do confirm the importance of post-RGD helicity for highaffinity binding to αvβ6, as 10 μM A20 DV1217 was required in order toobtain a similar degree of binding as 10 nM A20FMDV2. In this assaytherefore, loss of helicity resulted in a 1000-fold loss of anti-αvβ6potency.

Rational Design of Disulphide-Cyclic Derivatives of A20FMDV2

We though that as linear peptides may sometimes be susceptible to attackby serum proteases in vivo, that the cyclisation and use of D-aminoacids could be investigated to stabilise the peptides while maintaining,or improving, their biological activity (Okarvi, 2004). Rational,structure-guided design was therefore used to derive twodisulphide-cyclised variants of lead peptide A20FMDV2. The aim wasthree-fold: to stabilise the active structure, thereby increasing theaffinity; to improve resistance to serum proteases; and to introducesuitably positioned lysine and tyrosine residues to allow directradiolabelling with 4-[¹⁸F]-fluorobenzoic acid (¹⁸F-FBA) or ¹²⁵I,respectively.

Disulphide By Design software (Dombkowski, 2003; www.ehscenter.org/dbd/)was used with the solution structure of A20FMDV2 in 30% TFE to identifya pair of residues which were considered to meet spatial and geometricalspecifications for possible replacement with disulphide-bonded cysteineresidues. Lysine and tyrosine residues were added for radiolabelling.However, in order to maintain the entire structural unit of A20FMDV2 andprevent possible interference with the αvβ6-binding activity, theseresidues were added at the N-terminus of the peptide as a D-amino acid‘tail’. This peptide was designated DBD1 (see the table below).Ironically, preliminary serum stability studies indicated that theD-amino acid ‘tail’ may itself be susceptible to proteolysis. PeptideDBD2 was therefore designed in which all residues are contained withinthe disulphide ring (Table 6.1). Peptide ‘Ran’ was synthesised as acontrol and consists of the same residues as DBD1; however the residueswithin the disulphide ring have been scrambled.

In order to allow direct analysis of peptide binding to cellular andrecombinant αvβ6, a biotin moiety and spacer was also added to theN-terminus of each peptide (biotinylated-A20FMDV2, -DBD1, -Ran and -DBD2are thus referred to as B-A20FNDV2, B-DBD1, B-Ran and B-DBD2).

TABLE 8 Sequences of cyclic and control peptidesResidues in lower case represent D-amino acids. The RGDLXXL (SEQ ID NO: 1) motif is underlined. Cysteine residuesused for cyclisation are highlighted inbold and are underlined. Tyrosine (y/Y)  and lysine (k/K) residues added to en-able direct radiolabelling with ¹²⁵I and  ¹⁸F-Fluorobenzoic acid respectively.Glutamic acid (e) and lysine (K) residuesadded to potentially enable side-chain-to-side-chain covalent cyclisation. Peptide Sequence (SEQ ID NO)Modifications A20FMDV2 NAVPNLRGDLQVLAQKVART None (8) DBD1eykCPNLRGDLQVLAQKVCRTK Disulphide- (14) cyclised RaneykCKLVGALQPDNVLQRCRTK Disulphide- (15) cyclised DBD2CYVPNLRGDLQVLAQKVAKC Disulphide- (16) cyclised

Affinity and Specificity of Cyclic Peptides in vitro

Affinity of the cyclic peptides for αvβ6 was first tested in anon-competitive binding ELISA. Biotinylated peptides were allowed tobind rsαvβ6 immobilised on ELISA plates and bound peptide detected withperoxidase-conjugated ExtrAvidin. The scrambled peptide B-Ran did notshow any binding, but both B-DBD1 and B-DBD2 showedconcentration-dependent binding to rsαvβ6. Levels of binding weresimilar to those of B-A20FMDV2. Quantitation of the data by fitting of adose-response curve and subsequent calculation of the peptideconcentrations required for 50% binding (EC50) demonstrated thatB-A20FMDV2, B-DBD1 and B-DBD2 exhibit similar levels of potency in thisassay, consistently showing detectable binding at low nanomolarconcentrations (see Table below).

TABLE 9 EC50s for binding of biotinylated cyclic peptides to immobilisedrsαvβ6. Data were fitted to a sigmoidal dose-response curve and thepeptide concentration required for 50% maximal binding (EC50) determinedfor each peptide. Data represent the mean and standard deviation of theEC50s from four independent experiments. Mean EC50 Standard Peptide (nM)Deviation B-Ran ND ND B-A20FMDV2 1.20 0.28 B-DBD1 0.69 0.18 B-DBD2 1.701.16 ND, not determined.

Peptide specificity for αvβ6 was assessed by comparison of binding topaired αvβ6-positive and αvβ6-negative cell lines A375Pβ6 and A375Ppuro(FIGS. 9 and 10). Both cell lines express integrins αvβ3, αvβ5, αvβ8 andα5β1 at comparable levels, however only A375Pβ6 expresses αvβ6. Bindingof biotinylated peptides was assessed by flow cytometry. B-A20FMDV2,B-DBD1 and B-DBD2 showed concentration-dependent binding to A375Pβ6,with high levels of binding at concentrations as low as 1 nM. Incontrast, these three peptides exhibited only low levels of binding toA375Ppuro, and then only at high concentrations. The control scrambledpeptide (B-Ran) did not bind to either cell line.

In order to confirm the specificity of the interaction with A375Pβ6,binding of 1 nM peptide was assessed in the presence of either 63G9, anαvβ6-specific function-blocking monoclonal antibody, or an irrelevantIgG control. B-A20FMDV2, B-DBD1 and B-DBD2 bound strongly in thepresence of control IgG; however in the presence of 63G9, binding wasgreatly reduced, and in the case of B-A20FMDV2 and B-DBD2, completelyabolished. B-Ran did not bind in the presence of either antibody. Theresults confirm that at 1 nM, B-A20FMDV2, B-DBD1 and B-DBD2 bind toA375Pβ6 primarily through αvβ6. Thus B-A20FMDV2, B-DB1 and B-DBD2 haveboth high affinity and high specificity for αvβ6 over αvβ3, αvβ5, αvβ8and α5β1. In addition, peptide binding is stable and long-lived, as thepeptide-integrin complexes are stable to repeated treatment with EDTA.

In vivo and in vitro Studies with ¹⁸F-labelled A20FMDV2 and DBD2

Lead peptides B-A20FMDV2 and B-DBD2 therefore exhibit high affinity andhigh specificity for αvβ6 in vitro. A20FMDV2 and DBD2 can beradiolabelled at the N-terminus of the peptide to generate ¹⁸F-A20FMDV2and ¹⁸F-DBD2). The potential use of integrin αvβ6 for imaging andtargeting purposes can be assessed by injection of labelled peptides(with F¹⁸ or other radioactive moiety) into mice bearing pairedαvβ6-positive (DX3β6) and αvβ6-negative (DX3puro) xenografts to allowspecific visualisation of the αvβ6-positive tumours.

Discussion

The integrin αvβ6 is a major new target for the imaging and therapy ofcancer. As a step toward creating anti-αvβ6 reagents we used a rationaldesign approach based on known ligands of αvβ6 to generate peptideantagonists to αvβ6. These studies have revealed the structural basis ofnovel integrin-ligand interactions that are important for the biologicalbehaviour of αvβ6. We first noted that the potency of peptideantagonists to αvβ6 increased with increasing length of peptidesuggesting secondary structure in these linear peptides. The possibilitythat our peptides may have a helical motif was based on the crystalstructure of FMDV (Logan et al 1993). These authors showed that the G-Hloop of the VP1 capsid protein of FMDV consisted of an RGD motif at thetip of a hairpin turn followed by a 3₁₀ helix. This structure wasrevealed only if di-sulphide cysteine crosslinking between the VP1 andVP2 proteins was present. We examined the helical propensity of ourthree lead peptides A20 FMDV1, A20 FMDV2 and A20 LAP using AGADIRsoftware. The prediction was that there was an increasing helicalpropensity in the order A20FMDV<A20LAP <A20FMDV2, a sequence thatcorrelated with biological potency. Far UV/CD analysis confirmed thatall the 20 mers showed an increased helical nature upon addition of TFEfrom 0-50% (v/v). The wider profile for transition to the helical formfor A20FMDV suggests that a higher proportion of TFE is required withthis peptide to form a stable helix and that helical propensity of thispeptide is lower than for A20FMDV2 or A20LAP data that confirms theAGADIR prediction. The FarUV-CD data also was used to predict whatconcentration of TFE was needed to obtain comparative structures of allthree peptides by NMR. The mean elipticity plot suggested that at 40-50%TFE stabilization of the helix was forced to completion for all 3peptides. Thus a concentration of 30% (v/v) was used as it lay at theedge of transition for both A20FMDV2 and A20LAP and allowed fordifferences in helical propensity of the peptides to be revealed. (Toallow direct comparison, 30% TFE was also chosen for all subsequent NMRanalysis).

Structural assignment of all three peptides by NMR enabled theidentification of over 97% of all resonances with the majority of absentresonances from Thr2 of A20FMDV1 and Gly1 from A20LAP being difficult toassign as a result of amide hydrogen exchange and overlap. The highdegree of assignment enabled precise contact assignments for structureelucidation of each peptide and the evaluation and documentation of keycontacts involved in the formation of α-helices as shown in FIG. 3 andFIG. 4. Contacts shown in FIG. 3( a) for A20FMDV highlight that thishelix is the least defined under the conditions used. aH-NH i−i+3 andaH-bH i−i+3 are not continuously defined throughout the regionC-terminal to RGD and the helical stretch from Ala10-Thr14 is notdefined with hydrogen bond acceptors and f restraints. In contrast,A20FMDV2 contacts as shown in FIG. 3( b) highlight a well formed helixfrom Leu10-Val17 with aH-NH i−i+3, aH-bH i−i+3, NH-NH i−i+l and hydrogenbond and f restraints. A20LAP constraints in FIG. 3( c) falls in betweenthose observed for A20FMDV and A20FMDV2. Over the helical region ofLeu10-Gly15, A20LAP has a high degree of aH-NH i−i+3 contacts definedbut a poorer number of aH-bH i−i+3 contacts defined. Also in A20LAP, thehydrogen bond and f restraints are better defined at the N-terminal endof the helix but are absent in the C-terminal section. The shortfall inthe defined hydrogen bond and f restraints for both A20FMDV1 and A20LAPhave contributed to the reduction in helix formation in the NMR datacreated models but reflects the fundamental differences between thesepeptides in 30% TFE (v/v). The scale of helicity afforded from thecontact data where ideal helicity is observed by A20FMDV2, with A20LAPsomewhat less ideal and A20FMDV1 being poor can also be seen directlyfrom the experimental data as shown in FIG. 4. A20FMDV2 data in FIGS. 4(b), 4(e) and (4 h) shows more contacts and higher dispersion of signalsthat are indicative of structure being present. Once again, theseobservations are reduced in A20LAP with the number of contacts anddispersion being the lowest in A20FMDV. Contact data from Table 2confirms these visual observations. Regardless of the nature of thesehelices, it is clear that each peptide adopts a turn conformation andthat long distance contacts (i.e. between residues in the N- andC-terminal halves of the peptides) are observed in all peptides. Thesecontacts are most numerous and well defined in A20FMDV2 and suggest thathelix formation is key to forming a stable turn conformation. However,even though the N-terminal 6-7 amino acids appear not to have structure,they may still be important from activity considerations since there area number of NOE interactions between and N- and C-terminal residues thatlikely serve to stabilize the overall 3D structure.

The trend in overall helicity for each of these peptides(A20FMDV2>>A20LAP>>A20FMDV1) as outlined from the contact data isfurther supported upon structure elucidation using CNS software. Thestructural information has allowed quantitative analysis of the helicalpropensity of these peptides in a way that was not immediately clearfrom the FarUV-CD data presented in FIG. 1 and FIG. 2. Ensemble averagesin FIG. 5 show that for each peptide there is a helical section thatlies directly C-terminal to the RGD motif. The helix is shown to beapproximately 1.4, 1.6 and 2.2 turns for A20 FMDV1, A20 LAP and A20FMDV2 respectively and appear to agree with the trend observed fromAGADIR regarding the overall predicted helicities of these peptides. Thenature of the helix that forms directly following the RGD motif enablesthe side chains of the previously highlighted residues LXX[L/I] toprotrude from one side of the helix. As a result, this would create astructural motif involving a helix that is not dissimilar to the LXXLLmotif recently illustrated that binds peroxisome proliferator-activatorreceptor (PPAR) (Klien et al, 2005). The RGDLXXL (SEQ ID NO: 1) sequencewas identified as an αvβ6-specific motif by Kraft et al (1999) usingpeptide phage display and the importance of these residues wasdiscovered in earlier studies that examined the critical amino-acids inFMDV derived peptides that were required to inhibit experimentalinfection by FMDV (Mateu et al, 1996).

Our STDNMR investigation using A20FMDV2 peptide with integrin αvβ6enabled the confirmation that residues LXXL were important in ligandbinding to αvβ6. The STDNMR difference data shown in FIG. 6 highlightsthe importance of interactions through the Hd of residues Leu10 andLeu13 together with the absence of Hd of Leu6 highlights immediatelythat binding primarily involves the section of the peptide fromArg7-Thr20. This is confirmed by analysis of the STD amplificationfactor shown for each residue that also highlights that the primaryinterface occurs with residues Arg7, Leu10, Leu13, Lys16 and Val17. Thusour data provide a structural explanation for the discovery of RGDLXXL(SEQ ID NO: 1) as an αvβ6-specific ligand since the helix brings intojuxtaposition the non-contiguous Leu10 and Leu13 residues which theinteract with the αvβ6 surface in a linear fashion. The significance ofresidues Lys16 and Val17 in integrin αvβ6 recognition also requiresattention as this observation highlights the likely importance of anextended motif beyond RGDLXXL (SEQ ID NO: 1). Secondary elevatedinteractions are also observed for Asp9, Val12 and Thr20. Since the STDdata was obtained in a physiological buffer (PBS) without TFE, itsuggests strongly that A20FMDV2 binds as a helix to αvβ6. The primaryinterface residues occurring in steps of three amino acids illustratethe formation of a helix within A20FMDV2 during interaction with αvβ6that would enable all primary residue side chains to interact as oneface with the integrin target. Furthermore, it is possible that at leastthe N-terminal section of the helix between Leu10-Lys16 could adopt a3,10-helix structure due to the LXXLXXK regular pattern in agreementwith Logan et al (1993). Our data suggest that peptides specific tointegrin αvβ6 require an extended turn conformation with an RGDLXXL (SEQID NO: 1) based motif. In addition to the immediate importance of thismotif, αvβ6 specific peptides require increased helical propensity andthe ability to form helices with increasing numbers of residuesC-terminal to RGD will bind with greater efficacy. This was, perhaps, anunexpected finding since development of peptide inhibitors to otherintegrins such as αvβ3 and αIIbb3 have often striven for the smallestpossible cyclic peptide. The α-helix motif for αvβ6 appears to haveseveral roles. Primarily, it allows correct orientation of the LXXL toenable hydrophobic side chains to interact with a binding site on αvβ6,but in addition it promotes binding by also presenting contact residuesin positions YY in an extended sequence RGDLXXLXXYY (SEQ ID NO: 17).Moreover, the long range contacts between residues in the helix andresidues in the N-terminus stabilize the hairpin structure and thuspresent the RGD motif favourably.

The combination of structural (NMR and far UV/CD analysis) andfunctional (ELISA and adhesion assays) data predicted that our peptidesantagonists assumed a helical component when they interacted with αvβ6.This was confirmed for A20 FMDV2/αvβ6 interaction by STDNMR. Theimportance of the helix in peptide binding to αvβ6 was shown byconservatively destroying the helix by replacing valines in the helixwith their D-isomers. The resultant DV1217 peptide had no helicalpropensity and a 20-40 fold reduced potency as an αvβ6 inhibitor.

Some substrates, such as fibronectin, are not predicted to have anα-helix C-terminal to RGD but can function as ligands for αvβ6. However,αvβ6 has a much greater affinity of binding for LAP than forfibronectin. Since LAP possess an RGD-α-helix motif our results offer astructural explanation for this increased affinity since, presumably,there are more physical interactions between αvβ6 and LAP than with αvβ6and fibronectin. Our data may also explain how αvβ6 can activate TGFβ.Thus activation of TGFβ1 (and presumably TGFβ3) by αvβ6 requires afunctional actin cytoskeleton possibly suggesting that physical tensionmust be applied to the TGFβ-propeptide, LAP. The large number of contactsites that occur C-terminal to the RGD binding motif in our peptidesoffer an explanation as to how this strong binding to LAP could bemediated. This may be αvβ6 activation of TGFβ through strong,helix-mediated binding, involving traction/tension, or possibly thebinding-induced stabilisation from unstructured loop to helix cause aconformational change in the LAP that releases TGFβ.

The RGDLXXL (SEQ ID NO: 1) motif is found in many proteins not all ofwhich are extracellular proteins. Based on these investigations it maybe suggested that new, yet uncharacterised, ligands exist for αvβ6,which may include, for example, rhesus macaque pulmonary surfactantassociated protein C. The presence of intracellular proteins withRGDLXXL (SEQ ID NO: 1) motifs may suggest that they may bind tointracellular αvβ6, which might be of biological use.

In summary, the 20 mer peptide A20 FMDV2 forms an α-helix C-terminal toRGD when it associates with the integrin. Since there is a correlationbetween helical-propensity and peptide efficacy, this suggests thathelix formation is not a consequence of binding to αvβ6 but rather thatthe ligand (A20FMDV2) must assume an α-helix C-terminal to RGD beforebinding and that this binding is likely to stabilize the helix. A majorfunction of the helix is to allow non-contiguous residues C-terminal tothe RGD motif to be presented as a linear face to the surface of αvβ6thereby increasing the potential contact points between the ligand andthe integrin. These data will serve as a structural framework upon whichto design potent αvβ6-specific reagents that will be required for theimaging and therapy of cancer as well as the treatment of some fibroticdiseases.

REFERENCES

All publications, patent and patent applications cited herein or filedwith this application, including references filed as part of anInformation Disclosure Statement are incorporated by reference in theirentirety.

Brunger et al, (1998) Acta Crystallogr. D Biol. Crystallogr., 54 (Pt 5),905-921.

Cavanagh, J., Fairbrother, W. J., Palmer, A. G., and Skelton, N. J.(1996) Protein NMR Spectroscopy: Principles and Practice, AcademicPress, London.

Delaglio et al, (1995) J. Biomol. NMR 6, 277-293.

Forood et al, (1993) “Stabilization of α-helical structures in shortpeptides via end capping.” Proc. Natl Acad. Sci. 90: 838-842.

Guex & Peitsch, (1997) Electrophoresis 18, 2714-2723.

Johnson & Blevins, (1994) Journal of Biomolecular NMR 4, 603-614.

Klein et al, (2005). J. Biol. Chem. 280, 5682-5692.

Koradi et al, (1996) J. Mol. Graph. 14, 51-55.

Khandelwal et al, Eur. J. Biochem. 264, 468-478.

Laskowski et al, (1996) J Biomol NMR 8, 477-486.

van Gunsteren et al, (1994) in Methods in Enzymology: Nuclear MagneticResonance (James, T. L., and Oppenheimer, N. J., eds) Vol. 239, pp.619-654, Academic Press, New York.

Yan et al, (2003). J. Magn. Reson. 163, 270-276.

TABLE 1 NMR assignment list of observed ¹H chemical shifts forA20FMDV-1, A20FMDV-2 and A20LAP peptides in PBS/30% (v/v) TFE at 10° C.All chemical shifts are referenced externally to a 100 μM solution ofdimethylsilapetane sulphonic acid (DSS) in PBS/30% (v/v) TFE. ResidueH^(N) H^(α) Others A20FMDV-1 (SEQ ID NO: 7) 1Tyr 8.336 4.341 H^(β2/β3)2.830; H^(δ2/δ3) 7.205; H^(ε1/ε2) 6.913 2Thr 3Ala 8.615 4.350 H^(β)1.522 4Ser 8.335 4.496 H^(β2/β3) 4.000, 3.920 5Ala 8.426 4.415 H^(β)1.500 6Arg 8.317 4.308 H^(β2/β3) 1.872, 1.969; H^(γ2/γ3) 1.706, 1.774;H^(δ2/δ3) 3.285 7Gly 8.407 4.007 8Asp 8.337 4.677 H^(β2/β3) 3.227, 3.3649Leu 8.245 4.286 H^(β2/β3) 1.782; H^(γ) 1.722; H^(δ1/δ2) 0.941, 0.99010Ala 8.235 4.394 H^(β) 1.328 11His 8.226 4.604 H^(β2/β3) 3.295, 3.394;H^(ε1) 7.275 12Leu 8.231 4.343 H^(β2/β3) 1.862; H^(γ) 1.695; H^(δ1/δ2)0.931, 0.970 13Thr 8.276 4.386 H^(β) 4.202; H^(γ2) 1.471 14Thr 8.1484.323 H^(β) 4.244; H^(γ2) 1.281 15Thr 8.246 4.347 H^(γ2) 1.474 16His8.312 4.762 H^(β2/β3) 3.225, 3.343; H^(ε1) 7.145 17Ala 8.506 4.425 H^(β)1.498 18Arg 8.278 4.392 H^(β2/β3) 1.838, 1.917; H^(γ2/γ3): 1.679, 1.742;H^(δ2/δ3) 3.325 19His 8.315 4.702 H^(β2/β3) 3.302; H^(ε1) 7.140 20Leu8.188 4.286 H^(β2/β3) 1.688, H^(γ) 1.688; H^(δ1/δ2) 0.941, 0.988A20FMDV-2 (SEQ ID NO: 8) 1Asn 4.115 H^(β2/β3) 2.912; H^(δ21/δ22) 6.912,7.622 2Ala 8.250 4.300 H^(β) 1.394 3Val 8.197 4.491 H^(β) 2.197;H^(γ1/γ2): 1.079 4Pro 4.459 H^(β2/β3) 1.961; H^(γ2/γ3): 2.124; H^(δ2/δ3)3.773, 3.920 5Asn 8.605 4.810 H^(β2/β3) 2.838, 3.001; H^(δ21/δ22) 6.743,7.776 6Leu 8.140 4.387 H^(β2/β3) 1.714; H^(γ) 1.714; H^(δ1/δ2) 0.906,0.946 7Arg 8.253 4.257 H^(β2/β3) 1.922, 1.997; H^(γ2/γ3) 1.717, 1.801;H^(δ2/δ3) 3.321 8Gly 8.272 3.982 9Asp 8.400 4.574 H^(β2/β3) 2.799 10Leu8.279 4.253 H^(β2/β3) 1.841, 1.896; H^(γ) 1.681; H^(δ1/δ2) 0.955, 1.01111Gln 8.065 4.123 H^(β2/β3) 2.452, 2.572; H^(γ2/γ3) 2.284; H^(ε21/ε22)6.866, 7.510 12Val 7.733 3.825 H^(β) 2.310; H^(γ1/γ2): 1.040, 1.15513Leu 7.987 4.149 H^(β2/β3) 1.773; H^(γ) 1.867; H^(δ1/δ2) 0.966 14Ala8.455 4.107 H^(β) 1.553 15Gln 7.823 4.192 H^(β2/β3) 2.517, 2.659;H^(γ2/γ3) 2.298; H^(ε21/ε22) 6.870, 7.515 16Lys 8.109 4.265 H^(β2/β3)1.748, 2.076; H^(γ2/γ3) 1.659; H^(δ2/δ3) 1.540; H^(ε2/ε3) 2.988 17Val8.282 4.012 H^(β) 2.250; H^(γ1/γ2): 1.025, 1.083 18Ala 8.030 4.367 H^(β)1.562 19Arg 7.946 4.516 H^(β2/β3) 1.927, 2.100; H^(γ2/γ3) 1.781, 1.849;H^(δ2/δ3) 3.283 20Thr 7.759 4.357 H^(β) 4.253; H^(γ2) 1.297 A20LAP (SEQID NO: 6) 1Gly 2Phe 8.276 4.576 H^(β2/β3) 3.114, 3.263; H^(δ2/δ3) 7.260;H^(ε1/ε2) 7.183; H^(ζ) 7.298 3Thr 8.208 4.407 H^(β) 4.236; H^(γ2) 1.1564Thr 8.110 4.314 H^(β) 4.256; H^(γ2) 1.239 5Gly 8.433 3.920 6Arg 8.2544.362 H^(β2/β3) 1.745, 1.868; H^(γ2/γ3) 1.614 H^(δ2/δ3) 3.166 7Arg 8.4614.237 H^(β2/β3) 1.797, 1.880; H^(γ2/γ3) 1.611, 1.686; H^(δ2/δ3) 3.2218Gly 8.042 4.257 9Asp 8.151 4.574 H^(β2/β3) 2.724 10Leu 8.144 4.174H^(β2/β3) 1.717; H^(γ) 1.569; H^(δ1/δ2) 0.851, 0.915 11Ala 8.176 4.204H^(β) 1.438 12Thr 7.880 4.188 H^(β) 4.281; H^(γ2) 1.188 13Ile 7.9203.984 H^(β) 1.829; H^(γ12/γ13)1.130 H^(γ2) 0.778; H^(δ1/δ2) 0.890 14His8.292 4.329 H^(β2/β3) 3.117, 3.274; H^(ε1) 7.295 15Gly 8.174 3.941 16Met8.139 4.457 H^(β2/β3) 2.007, 2.105; H^(γ2/γ3) 2.517, 2.601; H^(ε) 2.13017Asn 8.304 4.681 H^(β2/β3) 2.708, 2.773; H^(δ21/δ22) 6.915, 7.650 18Arg8.035 4.565 H^(β2/β3) 1.657, 1.761; H^(γ2/γ3) 1.483, 1.563; H^(δ2/δ3)3.097 19Pro 4.400 H^(β2/β3) 2.137; H^(γ2/γ3): 1.944; H^(δ2/δ3) 3.51520Phe 7.277 4.384 H^(β2/β3) 3.051, 3.133; H^(δ2/δ3) 7.310; H^(ε1/ε2)7.383; H^(ζ) 7.281

TABLE 2 List of NOE, hydrogen bond and torsion angle connectivities forA20FMDV-1, A20FMDV-2 and A20LAP peptides. A20FMDV-1 A20FMDV-2 A20LAPNOE's Intra- 17 39 41 residue Sequential 18 31 24 i-i + 2 16 35 36 i-i +3 12 32 26 i-j (>3) 10 40 23 Total 73 177 150 Hydrogen 3 8 3 Bond DonorsTortion φ 4 10 4 Angles

TABLE 3 Structural Statistics for 35 structure ensembles of A20FMDV-1,A20FMDV-2 and A20LAP peptides. A20FMDV-1 A20FMDV-2 A20LAP Backbone r.m.s0.65 0.59 0.63 deviation across the ensemble over six residues inclusiveof: DLXX(L/I)XX (Å) Energy contributions 0.18 ± 0.05 0.25 ± 0.06 0.20.0± 0.04   (kcal mol⁻¹) E_(NOE) 0.45 ± 0.06 0.91 ± 0.02 0.33 ± 0.04E_(dihedral)

TABLE 4 Amino Acid Sequence of peptides D-amino acids are shownin lower case and are highlighted in bold. All DBD1,DBD2 and Ran peptides contain a disulphide bond betweenthe two cysteines. Number of Series Name Sequence (SEQ ID NO) residuesInitial 7-12mers DD1                RRGDLATIH (9)  9 DD2           FTTGRRGDLATI (10) 12 DD3              TGRRGDLATI (11) 10 DD4              GRRGDLA (18)  7 DD5            FTTGRRGDL (19)  9 DD6              LRRGDRPSLRY (20) 11 DD7               LRRGDRPSL (21)  9DD8               LRRGDRP (22)  7 DD9             GGLRRGDRPSL (23) 11DD10             GGLRRGDRP (24)  9 DD11              GLRRGDRPSL (25) 10DD12                 RGDRPSL (26)  7 DD13             GGFRRGDRPSL (27)11 DD14             GSIYDGYYVFPY (28) 12 DD15            NAGRRGDLGSL (29) 11 DD16               GRRGDLGSL (30)  9DD17             NAGRRGDLGS (31) 10 DD18             NAGRRGDL (32)  8DD19             VPNLRGDLQVLA (12) 12 A20 series A20FMDV1           YTASARGDLAHLTTTHARHL (7) 20 A20LAP           GFTTGRRGDLATIHGMNRPF (6) 20 A20FMDV2           NAVPNLRGDLQVLAQKVART (8) 20 p18-INK series P_FMDV2           VPNLRGDLQVLAQKVARTLP (33) 20 P_18INK           SAAARGDLEQLTSLLQNNVN (34) 20 P_FMDV2-INK           VPNLRGDLQVLTSLLQNNVN (35) 20 P_INK-FMDV2           SAAARGDLEQLAQKVARTLP (36) 20 P_INK-FMDV2-X           SAAARGDLEQLRQKVARTLP (37) 20 P_INK-FMDV2-XX           SAAARGDLETLRQKVARTLP (38) 20 D-Valine A20DV12           NAVPNLRGDLQvLAQKVART (8) 20 peptides A20DV17           NAVPNLRGDLQVLAQKvART (8) 20 A20DV1217           NAVPNLRGDLQvLAQKvART (8) 20 Biotinylated B-A20FMDV1Biotin-εAhx-YTASARGDLAHLTTTHARHL (7) 20 peptides B-A20FMDV1-RanBiotin-εAhx-ARHALTYRTGATHLAHTDSL (39) 20 B-A20LAPBiotin-εAhx-GFTTGRRGDLATIHGMNRPF (6) 20 B-A20LAP-RanBiotin-εAhx-PGRTFHRFGMGAITRTGNDL (40) 20 B-A20FMDV2Biotin-εAhx-NAVPNLRGDLQVLAQKVART (8) 20 B-A20FMDV2-RanBiotin-εAhx-RQLNVDALNVAGVRALKPTQ (41) 20 1st generation DBD1            eykCPNLRGDLQVLAQKVCRTK (14) 22 cyclics B-DBD1Biotin-εAhx-eykCPNLRGDLQVLAQKVCRTK (14) 22 B-RanBiotin-εAhx-eykCKLVGALQPDNVLQRCRTK (15) 22 2nd generation DBD2            CYVPNLRGDLQVLAQKVAKC (16) 20 cyclic B-DBD2Biotin-εAhx-CYVPNLRGDLQVLAQKVAKC (16) 20

The invention claimed is:
 1. A peptide the amino acid sequence of whichconsists of B_(n)RGDLX⁵X⁶LX⁸X⁹X¹⁰Z_(m)X_(i), wherein LX⁵X⁶L has an alphahelical structure, B_(n) is a sequence of n amino acids each of which isany amino acid, X⁵, X⁶, X⁸, X⁹ and X¹⁰ each independently represent anyamino acid, each Z is a helix promoting residue independently selectedfrom the group consisting of Glu, Ala, Leu, Met, Gln, Lys, Arg, Val,Ile, Trp, Phe and Asp, X_(i), is a sequence of i amino acids each ofwhich is any amino acid, m is at least 1, n is 6 to 7, and n, m and iare selected to make the maximum length of said peptide 20 amino acids,said peptide having a modification selected from the group consistingof: (i) linkage to at least one non-peptide therapeutically activemoiety; (ii) linkage to at least one readily detectable moiety; (iii)linkage to at least one nanoparticle; (iv) linkage to at least onelipid-based vesicle; (v) linkage to at least one capping residue; and(vi) replacement of at least one amino acid residue of said peptide withat least one of a halide derivative thereof or a N or C alkylsubstituted form thereof or a combination of said derivative and saidsubstituted form.
 2. A peptide selected from the group consisting ofA20FMDV1 (SEQ ID NO: 7) and A20LAP (SEQ ID NO: 6).
 3. The peptideA20FMDV2(SEQ ID NO: 8).
 4. A peptide the amino acid sequence of whichconsists of B_(n)RGDLX⁵X⁶IX⁸X⁹X¹⁰Z_(m)X_(i), wherein LX⁵X⁶I has an alphahelical structure, B_(n) is a sequence of n amino acids each of which isany amino acid, X⁵, X⁶, X⁸, X⁹ and X¹⁰ each independently represent anyamino acid, each Z is a helix promoting residue independently selectedfrom the group consisting of Glu, Ala, Leu, Met, Gln, Lys, Arg, Val,Ile, Trp, Phe and Asp, X_(I) , is a sequence of i amino acids each ofwhich is any amino acid, m is at least 1, n is 6 to 7, and n, m, and iare selected to make the length of said peptide 20 amino acids, saidpeptide having a modification selected from the group consisting of: (i)linkage to at least one non-peptide therapeutically active moiety; (ii)linkage to at least one readily detectable moiety; (iii) linkage to atleast one nanoparticle; (iv) linkage to at least one lipid-basedvesicle; (v) linkage to at least one capping residue; and (vi)replacement of at least one amino acid residue of said peptide with atleast one of a halide derivative thereof or a N or C alkyl substitutedform thereof or a combination of said derivative and said substitutedform.
 5. The peptide of claim 1, 2, 3, or 4, wherein the peptide islinked to a non-peptide therapeutically active moiety.
 6. The peptide ofclaim 5, wherein the therapeutically active moiety is an anti-canceragent.
 7. The peptide of claim 1, 2, 3, or 4, wherein the peptide islinked to a readily detectable moiety.
 8. The peptide of claim 7,wherein the readily detectable moiety is a radioactive moiety.
 9. Apharmaceutical composition comprising the peptide of claim 1, 2, 3, or4, and a pharmaceutically acceptable carrier.
 10. A pharmaceuticalcomposition comprising the peptide of claim 5 and a pharmaceuticallyacceptable carrier.
 11. A pharmaceutical composition comprising thepeptide of claim 7 and a pharmaceutically acceptable carrier.